MOLECULAR GENETICS PCB 4522
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Date Created: 09/18/15
Eukaryotic Transc rip a amauiai Mogei ioitne PlC cm anNAPll ininitiation tiansaimion cvcie Hnlnenzvme vs inuitiaei assemblv Model for PIC structure Nme tnai RAPSEI shows siiniiainvto badevlal u iaaoi goinains 2 and 3 CTD of RNA pol ll large subunit i Tne carhoxyeterminal domain CTD ofme largest Subunit p n molng consists of an aria ofamino acid repeats 7 aa eacn ofme sequence T reSereProeThre SereProeSer 52re eatSln mammals 42in lants 25 in east 2 it mustne unphosphmylated forRNA poi ll to ioin tne PlC Becomes nyperpnospnoryiateo by TFllH Tnis process is linked to the transition between initiation and ion ation 3 Binds mediator complex T FllF RAPSD amp RAPM tetramer reduces nonspeci c DNA interactions ofpol involved in initiation and elongation decreases pausing and protects eiongation complex from arrest Basal factors TFllF RAPSD ampRAP74 Basal factors n c D Stimulation oi pnospnatase only occurs in the absence of binding to IIE after initiation eiongation TFIIE 34 amp 57 kDa subunits letramer 1 binds RNA pol II and recmils TIIH Basal factors TFIIH 9 sununits l closely associateo uitnTrllE pinoing ano regulation Basal factors kinase 4 functions in nucleotide excision repair NER 53 nelicase 3 prornoter clearance by cm activity 5 mutations can result in xenna pigm entosa in nurnans TFllH a supunits l TFIIH core 2 nelic TFIIK in yeast orCAK 3 suounits Phos hor cm ofRNA pol ll larg ano activates gc in gependentLinases CDKS cAllt gdk activating Klnase ases 3 to 5 He 2 grotein kinase complex Called lates the e Suhunlli Regulated lzvlne ch8 y nouuieoineuiaioi Basal factors ses ATP to 9 DNA me ica uire It th AK 3 suhunitsl CTD klrlase CUM n ycll Matl Note most mine OAK in lne oeii lsmassnclated Mn lFl H TFIIH involvement in nucleotide excision repair of DNA amp cell cycle a pnospnoiylates CTD g cAllt activity chlt activating klrlase activates cycllnrdependent klrlases CDKS Q preferential repair of template strano TFllEH functionsln transcription l pnospnor lates CTD Wnicn raciiitates prornot r clearance 2 togetherwlth TFllE E s using one of its nelicases 3 t0 5 Basal factors Basal factors TFllEH functions in transcription 3 required for formation of H uman TFII H EMderived structure Two Modelsfor the Activation of Transcription 1 Multistep Assembly Two Modelsfor the Activation of Transcription 1 Multistep Assembly Two Modelsfor the Activation of Transcription 1 Multistep Assembly Two Modelsfor the Activation of Transcription E D recruited separately Two Modelsfor the Activation of Transcription 2 Holoenzme Activator Nme ine neieenzwne model is laigelv discredited nnwduetnthe low abundance mneieenznne in cells and ine results ntChlP assavslnai snewmuilnaep recruitment events RNA pol ll exists in a complex with Mediator and TFIIF RNA pul ll 12 suhumts pan vaevvnasvnmvtmed cm a 2 ran pvuteins inai are invulved in laminating active and basaltvanscviptiun Mediatuvunlyattachestu ine hypuphusphuvylated CTD under phusphuvylated Transcrip Ian Cycle Transcrip ian Cycle Medlamr A pol llmedlalor lolnsme PlC lfme 5 unoey Phasphnvvlale D Building the preinitiation complex PIC R le 0quot TFIIH and TFIIF in binding RNA P 1 mm attaches to N erminus ofTFIEB Zn nger 2 RNA p011 attaches 8TB to 1mm core 121122 3 cm binds 113p RNA pol II with meidator plus TFIIFjoin quotmm the preihitiatioh complex RNApnlll TFIIE binds to TFIIF TFIIF also makes contact with TAF1 TAFS and TAF6 0 Note meulalmnnlshnwv TFIIE Building the preinltlatlon complex PIC Th a a h39quot em or RNA POI II to the TFIIE and TFIIH are prelmtlatloh complex TFIIH last to lo39quot the PIC39 TFIIE amp 1 TFiininds TFIIB nrterl ninlls Zn ngu 2 Carl erminzl dn a39 nflar e TFIIE attaches TFIIH to O submt MRNApnl II CTD bmdsTBP RNAWI u A poly era m odulates the actnnty of quot1quot quotmm W s 9 and a kinase cdk7 th posphorylates the cm o pol T Mme chmndleP TFIIE nm elm ave memalovano mematomm mm 5 Note w lmeladlnn Transcriptian Cycle Transcriptian Cycle mam luuses af mty fur b the CTD 3 art e CTD lS hyperphusphurylated Hyperphus CTD mslbuges frum TEF and mm Edlatur Note Cm phnsphnlylminn arts as Ihe agemhm ends me minquot phaseznd begins elnngminn Transcriptian Cycle Transcriptian Cycle CTD lS dephusphurylated by a husphatase aeuvny simulated by RNA b ears F TFHF m the absence ufTFHE ul H mmates and the prumuter vwm CTD hyperphusphurylated ane TFH TFHH FHA FHD and memalbnemam anbe pmmulev Rein t at an Transcrlptwn Cycle Scaffald camplex Terrmnatmn RNA pul HTFHF ebm legtlt TFHA Vurlknvskv Ram x amp Hahn CEBU Nammne 2257229 Veag nuc ea Ema Tem male m m nbmzed an m agnelm beads sq 15ca wM wmams mm m Mam mwana ms mmmmmmv mmnym Mdnuc ewdesvwz m immune mund ahmnscnman Sca um vmmsmmmmv mm mm mummamv mi N a mused massvcman mm m wdkmskv mg mmmmn mm m 225 m Raumces usaj nrlhe Eucam mums 1Ludevse a ZDDUTheubmumnrvemed BAG39W pvnvmesa um betweenme mn em avchapemnesHs Hsm anme mmeasnme 2 am Chem 275 66134617 2 Jemsch a Pvmwmams 2mm umqumn and n5 Km hnwdnse aveme vamuv hes Trends Cel m 1 335m 3 Mevsva 1996 Pvmenwm mechamsms and mndmns Plan Mal am 32 275732 Mussew Ree HuanE Fnedbevg a Juhndnn 1999 was vegmamrv mmp ex mm pvmeasnme mnclmns mdependemv mmmemvsws m nudemme emsmn vapaw Mal ceua 6877695 5 Guzman EaW Sun Pvakasm a Pvakash1995Veas1 DNA vepaw mmem RADZS pmmmes cummex vmmannn beMeen Uanswmmn mm mm and DNA damagevemgnmnn39admRADN 2 am Chem 27 838578388 uvas ma Mencwa a 5mm 2mm TAFcnmammg and YAFwdependem vmmsnmanscnpunnauvaawe rap 7 VWD suemm 12mm 7 m ahaum a Gveen mun Dmmd masses meaa Pmmmevs vevea ed bv dmeverma w vecvunmem mm 288 12m 266 Sample MSF file before modifying the title lines Note In addition to your unknown cDNA amino acid file you need to include at least three 3 additional amino acid FASTA files These should be from distantly related groups of organisms so that we can see domains that have be conserved over long evolutionary distances The conserved regions will correspond to functional domains gtgil21357189lreleP76495691l Mediator complex subunit 27 CG1245 PA Drosophila melanogaster Note this is the unknown MDKLNSTLTAVKNLRSNVRLCFEHLADGTDGESGEESRNKFVNDFQERFAAINSQIREVEQLINGLPVPP TPYSLGNTAYLAQETSQDRQALYPQLVNSYKWMDKVHDHSFLAFNNLNQNTLRRSYNYCSQKRQRLPFSS tNNDBDHlDKLL hlvlkk l lKIERfEUlN ll llSNVMKAAlVFKGVLIEWVTVKGFDEPLEHDD LWAESRYEVFRKVQDHAHSAMLHFFSPTLPDLAVKSYlTWLNSHVKLFLEPCKRCGKFVVNGLPPTWRDL RTLEPFHEDCRNC gtgil157116185 lreleP7001652785 1i mediator complex subunit putative Aedes aegypti MNLEPTNN T HLRMLR eo thlbbNb FOKFTOFTOFTTNGVNSNLREFETCINDLT PPQAPFNT NT YT TFTNTFRO TYPHLVQSYKWHDKTHFY TFASVTTOON TKR YYTNTKRRR TP SSHLATPQTVDNLIGSIHFPNMNLKIVRPFMTN TTHTTT R LRAAVTTKGTTTFWVTVKGFDF TTDG VDEHWTVSRHQVFRKVQDHAHSAMLHFFSPTLPDLAIRSFITWFRSYLTLFADPCKKCGKHLHNTLPPTW RDLRTLEPYHEECKQ gtgil28558979lreleP700426O2l mediator complex subunit 27 Homo sapiens MADVINVSVNLEAFSQAISAIQALRSSVSRVFDCLKDGMRNKETLEGREKAFIAHFQDNLHSVNRDLNEL T RTIVKGYNENVYTEDGKLDIWSKSNYQVFQKVTDHATTALLHYQLPQMPDVVVRSFMTWLRSYIKLFQAP CQRCGKFLQDGLPPTWRDFRTLEAFHDTCRQ gtgi l 17564368 l ref lNP75O5386 1i MeDiaTor family member mdt 27 Caenorhabditis elegans MQSNQQPTTSANASGPSGARISSGNIPHSQSMPVLQSGNTANRPSTAGNPAAGRPILTLPSNTTEKHVIA YGILEDMIIGGEDEDLYNQEQILHSKRKVYREFTKSAKEIILCSPVTKFTAPTNFAQCNNYIQSYVNCFS TKCYYCKKHLRQFMPPTVVTRESSPIICHKLCLMSQVP Molecular Genetics PCB4522 Spring 2004 Lecture 3 Replication part B Dr Eva Czarnecka Verner Course web page http PCB4522IFASUFL EDU or go to Microbiology 8 cell Science home page and look under course material The ReplicationReplicon part B Chapter 12 Genes VII Chapter 13 Genes VIII The RepliconReplication Plasmid incompatibility is connected to the regulation of copy number and segregation 1 Compatibility group a set ofplasmids that are unable to coexist in the same bacterial cell a Both plasmids have the same type of origin b Copy number is controlled repressor RNA 1 that measures the concentration of origins 2 ColEl plasmid as a negative control model for copy number and incompatibility system ca 20 copies per cell Sel sh plasmids with territorial rights Plasmid incompatibility is connected with copy number 1 Primer RNA 555 bases starts upstream of the ori and extends into the ori a cleaved by RNase H cum RNADNA hybrids b 3 OH of RNA serves as primer to initiate DNA synthesis 0 only cut by RNase H if it is not duplexed with antisense RNA RNA I Replic on of CoE1 plasmid DNAmulticopy control Replication starts with transcription RNA II is required for priming D synthesis RNA polymerase at the origin Positive regulation 555 20 RNA PIIEgt origin Pl and Pll promoters for the regulatory transcripts RNA I is complementary to the 5 terminal region ofRNA primer II Replication of CoE1 plasmid DNAmulticopy control RNAloop stmcture 39 d o if loops are ds stem ss loop Replication of ColE1 plasmid DNA Replication of ColE1 plasmid DNA multicopy control in l 39 u tlcopy control 555 265 20 555 265 20 pgt origin pgt origin RNA39 RNA ll rep Ica Ion Replicase RNAI h bri 39zes to the5 terminal re Ion of RNA ii and disrupts the RNA loops Re lication of ColE1 lasmid DNA p multicopy coztrol Plasmid incompatibility is connected with copy number 555 20 pgt High 1 RNA I antisense inhibitory transcript forms duplex With Primer RNA II lt ltZl Pl 3 No replication u 2 The Primer RNARNAI duplex molecule RNA quot transcrip 39on is not cut at ori by RNase H and the WW persistent hybrid at the origin is not formed 0 DNA synthes1s is not initiated g New incompatibility group Plasmid incompatibility is connected generated by mutations with copy number 4 Mutations in Primer RNARNA I interaction region Q2 HOW 1065 RNA I negative regulator counts may result in formation of a new compatibihm the copy number grou A PI promoter regulates the expression level of RNA I RNARNA duplex can not be formed between the regulatory transcript M1 W and the L g W The mutated and original replicons no longer regulate 1 At low levels of RNA 1 replication occurs each other Original amp mutant plasmids behave as members of 2 At higher levels of RNA 139 mplication Shut different compatibility groups down Plasmid incompatibility is connected origins 0f yeaSt replicons ARS with copy number autonomously replicating sequence 1 about 50 bp in length and very ATrich Q How many levels of regulation A Positive Primer RNA 11 enhances replication by 2 Domains M0 domains providing 3 OH end a A domain 14 bp 11 bp core ARS consensus Critical for ARS function Binding 39te forthe origin recognition complex ORC which contains 6 proteins B Negative RNA I rgpressor shuts down replication but allows continued transcription of RNA 11 b B Domain mutations reduce orifunction C Negative Rorn protein helps to shut down replication 1 several B elementsimperfect copies of the because it enhances RNA primerRNA I duplex core ARS consensus 911 formation 2 transcription factor binding sites The yeaSt ARS The ORC origin recognition The yeaSt ARS complex ORC Origin recognition com lex Transcription iactor ABFl 1 6 proteins 400000 Dalton 400kDa enhances ini39ja39jon 391 2 must bind ATP before it can bind to the RS core consensus E3 omain core consensus Origin can function core consensus effectively With B domains functional A imperfect consensus ORC binds to A and Bl m Events at A and dommn and any B1 critical for initiation two B Elements The yeast ARS Llcensmg factor conSlsts of MCM proteins Genes vn Chapmr 13 amp Genes vnr Chapter 14 E I m 0R 3 o c ound to an through cell cycle 5quot V AE1 protected against DNase hypersens I e site in E1 Cd Cdc6 synthesized in 51 highly 1 G unstable degraded by ubiquitina MCM277 proteins iorm a Gaprotein ring complex Halflife of lt5 min 2 ORC hydrolizes ATP and loads MCM onto DNA Mcm complex recruited to the 3 ORC identi es origin orreplication ror Cdc6 amp MCM Late 51 Cdc6 G ARS by Cdc6 in yeastthese proteins entierthe nucleus at 31 In 4 rereplicatiun animals Mcm protetn are always in the umpiex Cdc6 amp MCM control initiation and licensing nucleus but only bojnd during 61 Mcm 2 3 5 Licensing factor s phase DNA synthesis initiated Cdc6 amp Mcm pusnepucmm proteins displaced l gt mp Ex 6E3 Cdc6 rapidly degrade no reinitiation 52 g to replicate or not to replicate That is the question MCM needed for initiation amp elongation a may contribute to helicase activity to unwind DNA Licensing factor controls eukaryotic replication nes VII Chapter 13 amp Genes VIII Chapter 14 1 The eukaryotic genome tens oithousands oireplicons Each origin activated only once in a single cell division 2 A rate limiting factor involved Used up by the replication event at each ori 39 Mus berenewed after cell division to allow further replication at that origin 3 Eggerimenlal System Xenopus eggsr can replicate DNA in a nucleus injected into an egg devoid of original nucleus wi out any new gene expression Material with NA can be in the form of a sperm nucleus or an interphase nucleus Xenopus eggs as system to study eukaryotic DNA replication Remove Egg cell nucleus one quot quotd f DNA synthesis Second round or ltl 39 DNA synthesis Block protein New pruteln syntnesis synthesis leads D No further DNA gerrneabilizatiun uf svn esis nuclear membrane Llcenslng factormust enter nucleus but can not pass tne nuclear membrane Licensing factor controls eukaryotic replication One DNA replication licensing factor Egt GE inactivated o 0 Cell division f LlcenSlng factor can not entertne nucleus breakdown of nu clear membrane some new licensing factor enters the nucleus Licensing factor ensures that onl single round ofDNA replication ccurs Components ofthe licensing factor 1 In yeast MCMZ 3 5 protein complex Enters nucleus only during mitosis N In animal cells MCMZ 35 complex remains in the nucleus through the cell cycle MCM3 bound to DNA before replication but released after it Other component of LF able to enter only at mito Is may be necessary for MCMZ 3 5 to associate with DNA cdc6 2 Ubigu ation system mutations allow rereplication Licensing factor degraded after start of replication Mitochondrial origins l Purple sulpnurbacteria able to nandle oxygen gaye eginning to euxaryotic rnitocnondria 2 Mltocnondrla are not Sexuallytransmltted 3 We all nave maternal mltocnondna 4 now is rnitocnondnal dsDNA replicated2 a use different on to initiate replication of eacn DNA strand b replication of Hrstrand starts first a in a D loop new L strand produced c replication of Lastrand starts new Hrstrand produced wnen on on tne old L strand is exposed bytne replication is nxi D loops maintain mitochondrial origins enes VII chapter 12 Genes VIII Chapter 137 i Slngle D loop displacement loop opening ofsooesoo bp in rnarnrnalian rnitocnondria 2 retranymena rnitocnondrial DNA nas 6 D loops at a tirne rnany origins and plant cnloroplasts naye 2 2 Tne new snort strand is unstable degraded and rersyntneslzed to xeeptne loop open 3 starts by syntnesis ofsnort RNA pnrnerwnicn may be extended by DNA pol tnis is norrnal for all DNA syntnesis D loops maintain mitochondrial origins Origin un Replication of mammalian mitochondrial 0mm H strand DNA has separate origins foreach strand L strand 39 L stran D loop displaced L strand i5 Single stranded Synthesis ornew L stra nd Note origin rorL strand i5 on tne s ran 1 RNA pnmewnmemd byRNA The snort strand i5 unstable and polymerase L strand dlsplaced tum over 500600 bases N 3 end cutby a 3estrand speci c endonuclese at Specific slles 3 OH extended into DNA by DNA polymerase Replication of mammalian mitochondrial DNA has separate origins for each strand E E L L Q new HQ ran h t d OriginfDrHs l eLsra Synthesis or new H strand is dela ed relative El HEW L strand 1 shun RNA pnrner synthesized by pnrnase 339 OH exrmdedlntn DNA by pnlyrnerase Duplex clrcle Partlallyrepllcated amp QQ II F11VI Gaps ii i new strands are sealed D loops may be maintained at mitochondrial origins The displaced strand ofthe DIoop remains single stranded until the origin on the L strand is reached This second origin initiates H strand synthesis This illustrates the principle that an origin may be used to ps and ii a m m 2 r a u a r n o a 5 539 m o m o m o U o o rolling circle PhiX174 modes of replication Rede ned Origin apegtrconan An origin can be a sequence of DNAthat initiates DNA synthesis using one strand as a template The problem of linear replications Since the new strand is always synthesized in the i to 3 direction it is easy to finish the new strand by running off the end of the template but how does the polymerase initiate at the 3 end of the template strand DNA pol usually binds region that surrounds origin 3 5 The problem of linear replications Solutions 1 convert linear to circular or multimeric molecules rolling circles examples lambda 7t phage is circular an T4 phage multimeric The problem of linear replications Solutions 2 create unusual structure at the end example hairpin so there is no free end linear mitochondrial DNA of Paramecium crosslinks the ends The problem of linear replications Solutions 3 end may be variable example Eukaryotic chromosomes have short sequence repeats at the termini A separate mechanism adds or removes these repeats It is not necessary to replicate to the end as long as some of the repeaw are copied The problem of linear replications S olutions 4 protein intervenes O en used by viral nucleic acids that have proteins that are covalently linked to the 5 terminal base adenovirus DNA 80 kDa phage 129 DNA poliovirus RNA 22 amino acids Strand displacement Linear template Adenovirus 1 bottom strand is used as template and the top strand is displaced 2 top strand forms terminal duplex before initiating DNA synthesis Free single strand 5 5 5gt 3 3s 5a gt 5 3 Dupler Ongln formed 3 3x 5s by base pamng Replicated lndgmdently Strand displacement Linear template Adenovirus Terminal proteindCTPDNA polymerase complex binds to 5 end ofthe top strand of adenovirus DNA 2 3 OH of dCTP serves as a primer for DNA synthesis 3 New strand is covalently linked to the initiating dCTP 4 old TP is displaced by the new TP for each new replication cycle Fg1313715G VIII Strand displacement Linear template Adenovims 1 Terminal proteindCTP binds to 5 end ofthe top strand of adenovirus DNA between 9 8c 18 nucleotide 2 Host protein nuclear factor I essential for the initiation binds between 17 8c 48 nucleotide 3 Initiation complex forms between positions 9 and 48 at a xed distance from the actual DNA end N39Fl 5 We enfofewurej X174 phage There are two types ofDNA replication in E can 1 lt3wa pnage eacn strand synthesized separately unidirectional replication fork m Synthesis ofthe r Strand 0 form the doublerstranded rm Serves as a model for lagging Strand Synthesis b synthesis ottne r strand to torrn singlerslrands for packaging into pnage particles servers as a model for leading strand sysnlhesls 27 ONE origin ofbaclerlal enrornosornal replication botn s trands synthesized att e sarne lime bidirectional replication fork DX174 phage as a simple model for replication Rolllng magma c r 535m r O Ogt gt gt O strand Replicative form RF ds plasmid O strand packaged to form virion Rol mg circle rephcatton IS amode1 forlead gs an at es Chapter 22 Activating Transcription Text assignment Lewin Genes VIII pp 631 to 652 Domain structure of activators Transcriptional activation nrotirs Make contact wnh hasal lacinrs and racrun than In the Illrmllmllnn mm dnncnn Upstream element DNA Transcriptional activatorsDNA binding domains Zinc containing DNA binding domains 1Zinc ngers CysZHisZ coordinated by asingle zinc ion Zif2 s 2 Eimetal thiolate clusterwitn 2 zin i ns coordinated by c c 3 Nuclear receptors steroids is related hormones hind ligand to activ te 2 zinc nrodules one for MA interaction and the other for dim erization each with 4 cysteines lucocor coid receptor Transcriptional activators Zinc fingers zifzss coo rw s Annnarallel Islmnd and an mhellx F M E my tnraezrncnngasnumnine mm necuav mngv v Ronenvveavenmmrawmu 39quot quot39939 quot quotquotZ39n58 Transcriptional activators Zinc fingers M M SP1 and Zif268 Ammarallel lemnd and an nnan me Mniecuiai EiningV 1999 W 39 RnbenWeavei MdSiaWHiH major groove Transcriptional activators GAL4 DNA binding domain is a bimetal thialate cluster 2 zinc ions 5 cysteines short u helix me Mniecuiai aminov mew I 39 Ruben Weaver Mmrawmu Gm mm mus n GAL4 transactivator protein DNA binding domain he x GALA dim er bound to DNA bimetal thiolate cluster Leucine Zipper coiledcoil mumMun InleIIlInns Hydrophobic herlad repeal gbcgefg leu leu val ile ala Transcriptional activators Nuclear receptors steroid receptors 2 zinc modules nne hlnds DNA the other Imam m mmmamn mm module cnnlzns 1 zlnc Inn mm a memes nus hlnd Imam thornnnED In zuwae Innson In the nudms RubenWeaei movame Transcriptional activators DNA binding module From Moiecuiar Bioiogy 1999 by Robert Weaver McGraW Hiii mucocorticoid receptor dimer plus DNA Steroid receptors 0 cys or his 0 dlfferent DNA binding 6 a Glu DNA binding Specl clty of DNA recognition 5 estrogen resides in aa between the two His residues on the aineiix giucocorticoid Three zinc ngers t into the From Moiecuiar Bioiogy 1999 by major groove Weaver McGraW Hiii Histone1 DNA binding domain Helixturnhelix This type of BBB is found in many transactivators HSFs amp homeodomain Heat shock factor DNA binding domain Helixmrnhelix Triple helical cluster relnlnrced wilh p strands Homeodomain proteins Helixmrnhelix Amnwema nnmmyre rmlllnu nma mminn In me hnrmont gme uniea H nmendnm activators nllen Ennlrnl development 39 nrgzn39sms ranging lnnn insects In plants rmm Mniecuial amlngv 1999 W RnbenWeavei M laWHlH Basic leu e Zipper proteins hZIP Coiledcoil dim erimion domain plus a basic DNA interaction domain 6cm yezsl activator requires SAGA complex GCNA dimerplus DNA Basic Helixloophelix hHLH inunim In dn39umllatlnn m rruscle rials me Mniecuiai EmiDQV 1999 W Runenvveaven McGiaWHiH MV D transactlvator Domain structure of activators Transcriptional ac at nine s DNA binding 1 Q 1 cm ain DED O lrrEr Upstream element DNA Acidic activation domains I cluster ofnegative charges I bulky hydrophobic residues 7 phe ala leu GO 9 96E 9 E SSTP amphip an helix amphip an helix VP15 frnmlhe rmmrmllan virus Mares srrl39llex IS the inn cnaramnmu Swwg sm w W mmmmns mm gammy mm mmm mm m mnan mmmmnnmn m WWW m Wmquot mum Swwg sm w W mmmmns mm 2mm 0mman ereimnm Swwg sm w W mmmmns mm 2mm 0mman ereimnm W manm mnhnm mum xqmmmonwm n mmmmm v m m x mum mm nnynnn Thunnny whnmn m mmm Adam W mmmmns mm 2mm 0mman ereimnm Addmve mans mummy mnmnnmnnmm w mm mm mmm w mm mm Adam W mmmmns mm 2mm 0mman ereimnm Addmve mans nhq gmnv n WWW r mum g m mm mm Mndzs nume Fundmn 4 Mmuummw mama m c m mm mm n m mm Modes ofAclivalor Funclio hurmn Modes ofAclivalor Function hurmn Yaxmuxwen used 9 mm mm cancenmusesha x 2 m muck wsubmdmg Modes ofAclivalor Function cnrmlex insiem IhEy rmke cnnlacl wnh enzeiivmms which in mm reerun basal ta nrs c cacti vato rs CEPIp300 PCAF M L39A mp Modes ofAclivalor Function The vslrngen receplnr msi recruits the cap in the early surges nllrznscriplinn and later recruits the Medianquot coactivators Estrogen receptor functions with coactivators mini em pm quotI a m uEannnJ mmmm am in We 5 3m in P160 Nuclear receptor coactivators min Nw uvlmlmm newquot 141 5n hlndslheER 27 muncps and cum hind man um am magnum Marmarv mam am Esmgen Neawasnas am no mmmnr Nate Wmhasmsovm w saw snmem News mm m 31d menymsngersse smug Modes ofAclivalor Function mm Stemm Acnvamrs 1 Glucncnmcnl 1cm mlnualcnnlcnld 1mm anemeen mm and Frnu emna am all 1mm hnmnmmas and reenamze me smumee IGHCL uanswes are nannemmes Spacing metaquot es n1 elmem the 51mm 2 r sme sure hm ns nan sne s IGACCL head we had mt 39llnrs z Ihyrnm mm Imamln n 1wmuumme and 1mm and Brusrlalnnlc mm mm nemnms arm haundlrmls ecnenmne haquot swans IGACCI man s I me E Sreu c recnunmnn s In uenced hy swung hawem rqens 1m Rxnmnmnmmm 3 hr min um m 5hr RAR heaem tau nxnhaem mmsw h vnk JRandRA Modes ofAclivalor Function me mymm mt 39llnr 1m and me Minnie am rBE 39Ilnr 1mm hind me sum mmessm all e rlmrmler mhe absence mllgand mug m meneane names am Wlnrlhecnmrlnssnr and allnws hmeme m mammms SMRT corepressor A grobacterium tumefaciens A na rural example of genetic engineering Chapter 131315 Gene VIII The bacterial Ti plasmid causes crown gall disease in lanls Agrobacterium tumefaciens is a soil bacteria that contains p an s Thetumor inducing plasmid pTi contains a portion that is that nction in the plant to produce enzymes that synthesize unique products caiied opines conjugates of basic amino acids and upketoglutarate or pyruvate 2 causethe plant to synthesize hormones resulting in tumors Crown gall tumors a natural example of genetic engineering from Genes VIII b B Lewin Agrobacteriumplant interactions compound I a h a 39 Agrobacterium in at It woun 51 e transfers T NAto plant cell Agrobacterium in soil f gml admum use opines as nutrients lnl ec on succeeds only on wounded plants Interaction of Ti Plasmid DNA with the plant genome Genes required to breakdown ogines for use as a nutrient source are harbored on the Ti glasmid in addition to vir genes essential for the excision and transgort of the T NA to the wounded giant cell 200 kb transfer to the plant opine camabolism Ti plasmids can be classified according to the opines produced 1 Nopaline plasmids carry gene for synthesizing nopaline in the plant and for utilization catabolism in the bacteria Tumors can differentiate into shooty masses teratomas 2 Octopine plasm s carry genes 3 required to synthesize octopine in the plant and catabolism in the bacteria Tumors do not differentiate but remain as callus tissue Ti plasmids can be classified according to the opines produced 3 Agropine plasmid 39 carry genes for agropine synthesis and catab mors do not differentiate and die out In 4 Ri plasmids induce hairy root disease on some plants and crown gall on others have agropinetype genes and have segments from both nopaline and octopine plasmids Ti plasmids and the bacterial chromosome act in concert to transform the p an 1 Agrobacterium tumefaciens chromosomal genes cth chvB pscA required for initial binding ofthe bacterium to the plant cell and code for polysaccharide on bacterial cell surface 2 Virulence region vir carried on pTi but not in the transferred region TDNA Genes code for proteins that prepare the TDNA and the bacterium fortransfer 3 TDNA encodes genes for opine synthesis and for tumor production 4 oc opine catabolism genes carried on the pTi and allows the bacterium to utilize opines as nutrient Agrobacterium chromosom al DNA transfer to the plant 0 0 V m pTi sareinLhesameintgroup The vir region is responsible for the transfer of TDNA to the plant wounded plant cell 0 virA is the sensor O activated virG membr constitutive virG constitutivelinducibe Note activated virG posmve causes i s own promoter to have a new stan point with quotquot Ems increased activity smnz Sensmn G AA Wm aim AD CM vecewev dumah v resrlnnse radiumquot p aceMsvmuune 9 M Q h A Hiskmase J a W tvansmntev 5 ml damam m quotWV an em Innsdu lnn VirA autophosphorytates at a histidme residue and then G 53233 signal muiecuies 9A Plant ceii mermvane We activates the response regulator by trahsterhhg the ph phate to DNA a partate residue if the must sham dumam dumam receiver domain of VtrG Bacterial TwoComponent System g W box prumuters The vir region is responsible for the transfer of TDNA to the plant wounded plant cell virA is the sensor Ver A5 triggers 1s produced by bacterial wounded plant membme of Vir A cells phenolic compound virG activates VirA phosphorylates transcription virG which causes from other vir virG to become virG promoters activated virG is the effector Ralcheling Up vir Gene Expression in Aglabarruium tumeaciens Coiled 5 its dine Klnase Signal Transdumon mnnmn um mm RenoNnr dumam m 1M1 m2 39 39m i m nu w m EEHPE limit 539 qpmr mmm on m swarm mm The vir region is responsible for the transfer of TDNA to the wounded plant cell VirA 6 ver ver nienihran Binds endor ssDNA protein overdrive nuclease indin ATPr 39 protein binding NA Binds a D1ampD2 strand Note The virAvirG system is related to the EnzZOmpR system that responds to om osrnolarity in other bacteria Generation of the Tstrand Left Right Border Border 5b TDNA 5b DVERDRIVE enhancer fthe o transfer process virDvirC VirD nicks the lower strand T strand at the right border sequence and binds to the 5 end Fig 18 29 Genes VIII Generation of the Tstrand TDNA Synthesis orthe new strand displaces the old strand Tstrand virDvirC 1 Helicases unwind the T strand which is then coated by the virE protein 2 one T strand produced per cell Generation of the Tstrand Left Right border border Tstrand integration 1 TrDNA transfer resembles bacterial conjugation rVirB op eron genes homologous to bacterial in genes 2 TaDNA integration process is not known rProbably through nonhomologons recombination rTargel sites ATrrich aTaDNA expressed at im site oiintegration genes that control transformed state oithe plant amp genes tor the opine synthesis 3 The ability oiAgmbactm39um to transier TaDNA to the plant genome m es it an ideal vector tor the introduction oinew genes into plants MiniTi TDNA based vector for plants Disarmed vectors do not produce tumors can be used to regenerate normal plants containing the ioreign gene 1 Binary vector the virgenes required for mobilization and transfer to the plant reside on a W 2 consists of the right and left border seguences a selectable marker kanomycin resistance and a Qolylinker for insertion of a foreign gene m iniTi MiniTi TDNA based vector for plants modified Ti plasmid Vir bom basis of mobilization 3 weeks after inoculation using TDNA seedlings Harvest time with Dr Verner Transformation of Arabidopsis plants Dip oral buds in 1 ml of Agrobacterium culture for 5 to 15 Detergent added to min allow bacteria to in ltrate the oral eristem Transformation of Arabidopsis plants mu m BEIEI seeds per mam Germmate un quot kanamymn mates m se ect transfurmants mm ZDtransfurmed wants perp ant 1n day an seedbngs W9 CQq un my a 3m 1r A variety of reponer genes can be activated by TNT in Plant and Bacterial Sentinels Plants respond to buried ordnance and khe gradient of TNT in rne soil environm ental Animal Vector Systems Amma Vemm systems I Receptormediated gene transfer g Egt 31m targetpmtem fur circulgr receptur plasmid target DNA transfemn and N Conjugake glycupmtems used furhver transfurmanun DNA uptake by endocytos s nucleus II Virusmediated gene uptake 0 fem Q 3 Adenovirus circular infectious plasmid musDNA particle DNA Conjugate viral particle and DNA uptake by endocytosis nucleus Use OfViIIJS to aid DNA uptake Endocytosis coated pits on cytoplasmic surface clathrin forms brous network 13 W354 JA I mated pit Molecular 021151a1agy by Damell Ludish amp Balnmure 1m Use OfViIIJS to aid DNA uptake Endocytosis coated pits on cytoplasmic surface clathrin forms brous network Mu1ccu1m 0211 En Dameu Ludrsh amp Balnmure 1m Retroviral vectors encapsidation signal w L4H extended packaging LTR 1 POL ENV RTR Wildtype 7160 H R l gene Insert 7 lab RTR vecto r I U lIIIIIIIj I AG A G ALTR 7 GA POL ENV PUMA llA A defective w V In G G virion core protein Packaglng POL rev transcriptase integrase amp viral protease cell ENV envelope glycoproteins Retroviral vectors RNA D D G Reverse transcriptase DNA 33 vector Q Integration Host DNA Q D D Retroviral vectors 1 based on disabled murine retroviruses 2 strong advantage is ability to integrate 3 two component system a packaging cell all signals for encapsidation have been removed from DNA Can not create an infectious particle unless the viral vector is present b viral vector only encapsidation and replication functions have been retained 7 kb insert c producer cell line is a packaging cell line containing a retroviral vector Retroviral vectors gene insert 0 I m 3 retroviral vector packaging prodquot infectiuuS cell line can line particles G I expression lt2 endocytosis of foreign lt1 by gene target cells i tegrat n to chromosome Adenoviral vectors 1 Late genes WI ype E1 MLP 23 In 35kb l 4 E4 mm gamma lt5 Jab MLP AE3 4 E a inverted term rep em enczp sidatinn nd 293 cell raphoatmn MLP major 1ate promoter Adenoviral vectors 1 can accept only 65 kb 2 although wild type virus can integrate into a specific region of human chromosome 19 adenoviral vectors have not been shown to integrate Remains episomal and is eventually lost works best to transfer genes to lung tissue Has been shown to carry cystic fibrosis conductance regulator protein to respiratory epithelium 0 III Chimeric RNADNA oligomers can direct correction of mutations Can use either strand as a target r i OmethRNA 9quot Clamp region of homology 1 II uTGCGCS aacuuccucTAGCAcccccugggngT T GC T AAGGAGATCGTGGGGGACCCC J 5y Nonlranscribed strand DNA Kren Bandyopadhyay amp Steer Mar 1998 Nature Medicine A 285290 RNADNA hybrids can direct correction of mutations Ser G FactorIX coagulation gene Arg mutated TJTGCGCS azacuuccucT ccccuggggT T T CGCGC TG GAGATCGTGGGGGACCCCTJ 3 5 Kren Bandyopaolhyay amp Steer Mar 1998 DNA Nature Medicine 4 285290 RNADNA hybrids can direct correction of mutations aTGCGCtieacacuuc cucTnGCncccccuggg IIIII IIIIIIIIIIIllllllll PEpolyethylenimine rcooac TGT Ge onrcorooooonccccr 3 5 lactosylated Intravenous injection of rats Uptake monitored in liver Kren Bandyopaolhyay amp Steer Mar 1998 Nature Medicine 4 285290 RNADNA hybrids can direct correction of mutations Features of the RNADNA hybrid 1 RNA 2390methylated to protect against RNase H 2 GC clamps to stabilize duplex formation No ends for nucleases 3 RNADNA hybrid works much better than DNADNA for excision repair Application of gene therapy to enetic diseases 1 Over 4 000 genetic diseases have been identi ed but only a relatively small number have been determined to result in a defect in a singl g classical Mendelian inheritance patterns 2 Target tissues limited by current techniques and knowledge in cell biology Examples of tissues Where gene transfers have been successful include broblasts lymphocytes endothelial cells muscle cells hepatocytes keratinocytes an bone marrow precursor cells 3 sometimes regulation of expression in target tissue is very complex 3 globulin synthesis must be coordinated With an gobulin synthesis Safety amp Ethical Issues 1 contamination With competent viruses39 careful monitoring of producer cell lines N gene therapy modi cation of cells for treatment or prevention of disease Cells are removed from one individual modi ed and either replaced to same or another individual somatic cells g line cells L Enhancement engineering modi cation of somatic or germ line cells in Ways not related to eatment of disease height strength intelligence 7 Version 2 PCB4522 spring 2007 Exam 1 Review Questions Lecture 3 Conjugation l N E 4 V39 0 gt1 9 gt0 What is bacterial conjugation Is having the F plasmid the only means for conjugation to occur What is an episome In what two modes can it exist Name three ways in which the F plasmid can replicate and which origins of replication does it utilize in each of the three situations What process is encoded by the transfer region rm and trb genes Approximately how many genes are present in the transfer region Note in gure 132 in the text that the rm and trb genes are intermingled within the transfer region How is the F plasmid integrated into the chromosome homologous recombination transposition IS Differentiate between the following types of cells F F Hfr F What origin of replication does the F plasmid use in Hfr cells What is the function of sex pilus What protein constitutes the pilus What gene encodes pilin protein What is the largest transcript synthesized from the transfer region What are the regulatory genes that control the traYtraI transcription unit Which proteins form the pore complex At what site and how does the transfer of the F plasmid begin Which protein nicks oriT In which direction with respect to the transfer region does transfer proceed What is the role of TraY Can mob containing vector plasmids transfer without the help of another plasmid UI ON 00 O N O N N N L N 0 LA 0 LA Version 2 What is the role of a helper plasmid What is the function of TraI A nuclease amp helicase ATP hydrolysis What is the function of TraS and TraT What is the F prime F plasmid How is it generated Which protein attaches covalently to the 5 end of DNA during F plasmid transfer How long does it take to transfer the entire E coli chromosome Which transcript of the transfer region F plasmid has a polarity that is opposite to all of the others What is the function of TraM How about TraJ What keeps an F cell immune from further conjugation from other F cells Which proteins are involved How have some bacteriophage exploited the conjugation process By what two mechanisms is the F plasmid integrated into the host chromosome Why are recipient cells F often not converted to Fr after F plasmidmediated conjugation with an hfr cell By what process does the donor chromosomal DNA become a palt of the recipient chromosome after conjugation Which end of the DNA is transferred to the recipient cell Is single or double stranded DNA transferred during conjugation Does conjugation just involve DNA transfer or is it also a replication event What are the two constants in replication cycle How much time does each of these functions take What are multifork chromosomes Note Questions below are not covered in the Handouts but you are still responsible forthe material in the text Genes V section 1321 33 U 4 35 L O L 1 LA 00 LA 0 4 0 Version 2 What system ensures that duplicate plasmids are segregated to different daughter cells produced by division Partition system par What are the components of a typical segregation system ParA ATPase amp ParB proteins amp target site parS also IHF Which protein binds to parS A parS is the region of DNA that contains the IFH binding site anked by boxl and box sequences The proteins that bind at parS include ParB and IHFx and IIF see Figs 1336 and 1337 Which protein binds simultaneously to boxl and boxB A ParB Which protein is transiently associated with the partition complex and has ATPase activity A ParA What is the function of partition complex attaches DNA to a membrane and sites of attachment are segregated by septum Understand the concept of plasmids that synthesize longlived killer and short lived antidote Is the partition system associated with single copy or multicopy more than 10 copies plasmids Homologous Recombina rion Lewin chapter 15 Genes VIII Version 1 Evolution could not happen without genetic recombination When mutations occurred it would not be possible to separate favorable from unfavorable changes Ultimately the chromosome would accumulate so many deleterious mutations that it would fail to function Quotation from Lewin Genes VIII p 419 By shuffling genes recombination allows favorable and unfavorable mutations to be separated and tested as individual units in new assortmenm It provides a means of escape and spreading for favorable alleles and a means to eliminate an unfavorable allele without bringing down all the other genes with which it is associated This is the basis of natural selection Quotation from Lewin Genes VIII p 419 Types of recombination Homo1ogons or generalized in eokaryotes it occurs at meiosis in males an in females in the formation ofthe gametes Sitespeciric phage integration inversion of speci c regions ofthe bacterial chromosome Transp osio39on does not depend on sequence homology speci c enzymes involved Version 1 some ecumb quot 39mggg m M Proohose o e is heceu t has dooi caved hs chromosomes so Mm4 no 39es ufecich ore oreseht A chiasma farms or the Fig 1 3 Section 11n Therefure o shgie chaniem recombhotioh eveht coh ohiv pruduce 50 recomb hams in o ot iswest bocterioisvsterhs The Holliday Model Two possibie outcomes 17 patch recombthattoh 2e spitce recombination The outcome depends on the pattern at the ing39 17 2nd nick Vi same strand as 1st patch 27 2nd nick on dtffereht strand sphce Version 1 Th e Meselson Rudding Model Addresses prubiem at what making by ertep prucess 17 briuup turmdttuh Nat was ur base paring mud Jay 7 thh gapped DNA 27 end prudum stttt Ci HuiitdayJunmtun Requiremenfs for recombinafion 11den1 icalor very similar sequences in The crossover region 2 Complemenfary base pairing 6 dsDNA 3 Recombinafion enzymes 4 Heferoduplex formafion 5 The poinf af which Two dsDNAs are held fogefher by complemenfary base pairing 9 synapse Formation of Heteroduplex DNA Holliday Junctions mnzah on z isomenzaukm lt I 4 Version 1 resolution resolution 39 A v E Exchange f Patch of anking i sequences 7 A B A E Holliday Junctions Weaver R F m Moizcuiar Bioiogy 1999 Mc raeriii Migration of Holliday Junctions s c E M L 1 s A Molecular Basis for Recombination 1 The major enzyme complex required for recombination is RecBCD 2 RecBCD has 339 to 539 exonuclease activity and endonuc lease activity 3 RecBCD loads onto the DNA at an end or at a break in the double strand Version 1 chsi tes and Recombination Part I chi s 5 1 Rec recombination is stimulated by chsites GCTGGTGG asymmetric move into DNA creating a loop that its C 2 RecBCD s exonuclease activity degrades Re D chsi tes and Recombination Part II 3 serves as end fur meme musicquot mediated by RecA 3 RecBCD encounters chi site in correct orientation exonuclease activity is inhibited 4 Stable single stranded 3 end is formed by endonuclease cleavage by RecD 5 Recombination is promoted on the 539 side of the chsite kchCD nuclease rexnuclease rendunclease rnelicase After Recb leaves RecBC only acts as mi case VerSion 1 czfsites and Recombination Part III 5 ssDNA forms triple stranded helix the implications of a RecD mLItan Important Note RecD 9 exonLIclease activity 1 Three stages of participation of RecA in strand exchange 1 Presynapsis slow RecA and SSB coat ss 2 Synapsis fast alignment only of complementary sequences in ssDNA and dsDNAs that participate in strand exchange 3 Postsynapsis or strand exchange slow ssDNA replaces one strand in the dsDNA The two DNAs are intertwined in ajoint molecule intermediate Role of RecA protein in Recombination 1 The ssDNA must find complementary DNA 2 RecA binds Hie ssDNA 9 extended elix Wid Version 1 Role of RecA in assimilation of invading strands admiresquot quot 39 17 aggregates into iong tiiaments Witn 57 or derNA o RecA monomers per turn ot tiiament wnicn nas a deep groove containing tne DNA a nucieotides per ReCA monomer 27 DNA neid in form that is 15 times extended f normai B form one turn per 1E nt dsDNA contacts RecA aiong minor groove leaving its maJor groove accessibie for reaction Witn 2nd DNA moiecuie Sr firsi siep lS for Rem i0 blYld ihe ssDNA Then ihe dsDNA is incorporated into ihe structure Role of RecA in assimilation of invading strands sweetietree 37 Inte action of t e two bNAs lakes place in ihe tiiament groove Detore actuai strand excnange wnicn requires a nickr end At end ot tne reaction RecA is bound to dsDNA 4 Large amounts otATp are hydrolyzed during tne release of Rem from We du legtlt DNA ATP ma aci M an aiiosteric tasnion cnanging tne affiniiy ot RecA tor DNA nign affiniiy wnen ATP is present low affiniiy wnen absent 57 Initiai neterodupiex DNA may not be in piectonemic relationship In parat iemiCJoiN ihe two strands iie side by side Witnout iYiieNWiYiiYig 3 Helical ssDNA wRecA can pair wiTh dsDNA in iTs major groove To form a Triple sTranded heix Version 1 randed helix 39I 39 4 The sTrands of The quotinvadedquot dsDNA are displaced and can Then pair wiTh respecTive sTrands of The quotinvadingquot DNA 9 Holliday JuncTion 1 221 DemonsTraTion of RecAdependenT synopsis in vim 274 bp nserT of shared homology 0 I H QEWZIrIng of u no and re lar39 DNAs Weaver R F mMulecular Biulugy 1999 Mc5PaWrHlll DemonsTraTion of RecAdependenT synopsis in vim 1 Paranemic helix This linkage was easily desTroyed by incubaTing for 5 min aT 2 deg below The The melTing TemperaTure No WaTsonCrick base pairing involved 2 The minimum lengTh of The homologous region found To be aT leasT 50 bp 3 Nickng noT required To iniTiaTe synopsis in vifra Nicking required for sTrand exchange Demonstration of strand exchange between sscircular and dslinear DNA Nule ssDNA emailed me Rem prulem Postsynopsi Jew molecule intermediate Weaver u r mMolecular Emlogy 19W mammal Version 1 Branch Migration A er RecA Whales New J mg 1711 M l3 the endonuc lease mm we direct dweenun uf out We a F mummy My wamwmu The RLIVABC Proteins amp Migration of Holliday Junctions 5 Migration increases the length of heroduplexes allowing for isomerization of Holliday Junctions 6 RuvA binds amp sTabiIizes Holliday JuncTion 7 RuvB Then binds To RuvADNA complex Version 1 drive The migraTion of Holliday JuncTions 8 RuvAB and RUVB Mimr ATP hydrolysis G B 7 L 5 9 IsomerizaTion probably does noT require proTeins or energy 10 Rqu binds resolves Holliday JuncTions by cleaving The Two crossed sTrands 139 Rqu 10 Model of the interaction between RuvA and u Holliduy junction Version 1 2227 w M Ma en Id 13me mi mm wiwwm 2223 Model of the interaction between RuvAB and u Holliduy junction based wquot 1 W ommn on EM images 2231 Model of the interaction between Rqu at u Hoiiideyjunction 10 Rqu binds quotresolvesquot Holliday Juncfions by cleaving The Two crossed sfr ands 1 lRqu Verslon l Imporan Nofe Recombinafion is dependenf upon canfar mafion of Holliday Juncfion af The Time of cleavage A a F 3 mm la snlullun A Exchange 7 Pm of ankmg sequences 5 a Bacvzmal the steps m recombmanoh pathway w 1712 Addifional Nofes on Recombinafion 1 Operans recA recBCD ruvABC ruvABC are adjacenf buf C is Transcribed 39 39 I 39 39 2 ther r39ec pafhways can subsfifufe for some of The profeins described 3 RecF pafhway amp RecG 4 Phage o en encode Their own recombinafion funcfions Vers on 1 Back To Eukar yofes ep a ene zygm ene RA om gra em mva ved mgg och Stages of Meiotic Prophuse mums u cme n n mamomm Early stages of meiosis in human fetal ovaries memmuwurs m Mlin amp Woman 2am J Version 1 memmuwurs m Mlin amp Woman 2am Central element in u recombination nodule synupionemul complex Eueanyoies Reeombinaiion is iniiiaied ai double sinand breaks Version 1 n m Wm EueanyoiesReeombinaiion isiniiiaied aidoubie sinand breaks n RadEO inyeasi sneaies 3 ss ends Campiex of prmeins waived in making aaubie sinana breaks no ms WWW quotMmquot 39 AVVAM sjxwib Spell is eovaieniiy joined io 539 ends of double sinand breaks Spell revers biy assuciclves wiih M2 3 Vvv enas 1i maybe a respuns bie fur cremmg m w Me n cks sowWW L Rad50 nexi creme 3 ss ends by exanusiease aeiwny 31quot c Fig M X Rad50 Human Dmcl bent at a Moments Khebuohtet at Mutecutar Cett200414 3637374 Verston t Ktnebuoht et at that Cett 200414 3637374 Recombination in eucaryo res 17 Doubte strand breaks occur ttrst Thts tdea ts w form synaptonemat comptexes Tradtttonat vtew was that the synaptottemat comptex was set up to attow recombtnatton 27 thj mutants do not form a synaptonemat comptex and shows no crossover tnterterence Thts suggests that the synaptonemat comptex spreads from the stte of the ds break and prevents further recombmattott events 37 Mutants that btoch axtat etement formatton do not btock ds breaks but do prevent recombmattott Recombination in eucar yo i es 4 The specificify of chromosome pairing is confrolled by Hop2 profein HopZmufanfs show ormal amounfs of synapfonemal complexing buf nonhomologous chromosomes are paired This suggesfs fhaf homologous pairing is nof required for synapfonemal complex formafion 5 ther profeins involved include MSH4 mismafch repair and Dmcl and Rad51homologs of RecA 6 Only a minorify of inferacfions resul139 in crossovers crossover confrol Meiosis is blocked if no crossover occurs To ensure fhaf cells do n f segregafe unfil crossover has occurre Version 1 17 Version 3 Bac rer ial Transcriptional Regulation Genes VII amp V chapter 9 Transcription Genes VI chapter 11 Transcription Transcriptional regulation in bacteria Subunit structure of bacterial RNA polymerase 160 kDa 3 150 8 3 70 O o 40 Oil iziiii W lthkLll I iiiiiiiiis i i i emu kl immnw aim iiii ll 5 ruli aiwiiie 105 km m lU Fig 916 Which subunits form the catalytic center Which is the largest subunit What is the mass of E coli holoenzyme amu ii l p i ii to 3 and makes a copy of maxi oiiiiiiiis mum umer Which subunit is required the template strand ofthe mm H mu for promoter speci city DNA ima kLii kt tiiigiyiie elmtr D What does the rpoD gene Wm WM Coding strand has the Luc H mmHi encode poAy poB and i J same sequence as the rpoC RNA Fig 9 3 RNA polymerase surrounds the bubble During transcription As the transcription bubble moves thelranscrlptlon along the DNA template the RNA is bUbble remalns displaced 39om the RNADNA hybrid Direction oftranscription inside the RNA polymerase which maintains the correct Enzyme movement DNA coding siiand Fe i alignments between thetemplate andthe Transcription l4 1225 m I 39 39 bubble Coding strand active site DNA 1 The transcription bubble is 39om 1225 upstream bases and the AIDNA hybrid is 89 bp in length Fig 95 5 downstream tem pl ate stran d nimiin Mmergituw Theigt i f RNA sygthes39s RNA is synthesized from 5 to 3 The incoming nt loses the gamma 395 quot Per 59 quot and beta phosphate groups leaving the alpha phosphate Version 3 Coding strand template strand ribosomes l5 aaSec 45 l llSec Tne rate ortranscnption is 40 ntsecondi wnicn is aboutthe s me rate as translation is amino acids second 45 nt or RNA second Transcription is mucn slowertnan DNA syntnesis 800 bpsec The transcription reaction has three stages ie template recognition closed promoter complex open promotercomplex 2e initiation 3e elongation 4e termination only need sigma nere Distribution or RNA polymerase Conservation in RNA polymerase structure polymerase Setn Darst Lab webpage Rockefeller University nttp Wyva rockefeller edulabneadsdarststructures ntm T1 RNA polymerase has a single subunll e e i Thumt Speci city is sheel binds in wlde groovet Qt 6V7 V while positions 1to t k a i ioentertne actlve quottl jalm t e e Enzyme movement 1 viiiiaiiax wtwergilom Fig 97 i Ixxcmesiie39 V i Finger T7 RNA era has a specificity loop that osi I 7 to T7 RNA polymerase r Asingle polypeptide cnain r omologous to DNA poll w ii ia all as Moyes rastertnan E col RN polymerase 2oo ntsec vllrirl quot DNAlleSll lthe palm T n rorDN is bout25Aarid isconseryedi all A 1 polymerase l l i6 nt in bacteria and 25 nt in eukaryotes Structure of the transcribing RNA polymerase The 17 RNA Fig i structure or transcribing T7 RNA pulymerase Vers on 3 Structure oflhe transcribing RNA W DNA 5 mead m make a mm mm W aHhE acme sne by aWaH u olymer pvmem Twashaypbene E quotchina mm mm hexpsmp me R NAawayhum m WW We temmate wand The new eunnguansen n quotNquot W e h mm a a A mm mm 5 23m 23m Enzyme suuduve pvuwdes mswgm mm huwms makmg and bvea Mcumad u M39W OnethedHemmasuVany nuc ewcamdpu ymevasewstha J 39 kmg scan ccuvA he enzyme mus make new pvmem budge s1vuctuve appeavs tuchangecunmv Wm l V V 7 p y untamwnhthenew ya ed nudeum as m bveaks cumad LELLLA I x OEMUM W h he 511 d AIM WWW 2aag ssnizg msg a Emp a gm m 1 WWW w MM H mm nudeasme enzyme muves quotWWW quot 2 p p F 7 mm pusnmmuvm addnmn u r quot 39 henex nudeuudeand L LLEEL e vee h cumamwnmhe r tam eymmax dumam Magma 7n 5 ma a m We acme 5N2 uvRNApuL mwmmkmg mevacuun Wm DNA he nEen gmmmev cumg ex s a a unmn uHhe N4evm u um N Mhemhu uenzyme MENr 39 Yhe DNA channe VVhent Version 3 imam am ii n m new Mia nun amnesia main um 3 min mania inn mums m cm 3 ng an aim imaiaiama a ii mmquot Bacterial promoters 1 Core promoter 10 and lt15 eIem ents 10 TA AT lt15 TTGACa ting seque 2 Upstream activa nces subunit of RNA pol UP binds alpha quotDwain aiimimi Sequence 1 III II I LIP SD in as in 1 pissie uyeienen caienanmg iysinaieasei iisui rmB operon irihnsnmzl RNAsi Differences between RNA polymerase and DNA polymerase Transcription cycle z RNAPscan menine DNA uu iex 3 R NAPs iniiaiiun is piimeu by a singie nucieulide nui an uiigu as is ine case iuiDNAPs R NAPs make muilipie cumamswilh ineztoH uiine incummg NTP 5 DNAsciunching accuisiui R NAPs aiiuwmg abullive cycimg wniie siii lelaimng canted Win ine plumulel FDVR NAPs lheliansciipl is peeieu awayiiumine lempiale nui su Ahnniv nn Ell cycimg R NApuiliansciibe 279 ni anu in ni i n n n ii ine plumulel May DEEUY seyeiai AA nunuieuiimesbeiuieiiue u 7quot eiungaliun niinaiinimkfg 39w m Nuie inenumbeiuibasesinaican M rj V iiiquot be packed min ine active siie mm Wquot enzyme i539 T is Dune aiesWiin myiin swam abumve piuduclsui ri bases MW new V l me 7 iniiiaiiun uisyninesis is ieguialed by many piuieinsiui R NAPs but nuiiui DNAPs Wnai is ine iasiesi plumulel ieavancelime7Which piumuieiv Fiu 919 Transcription cycle Fuvmaliun uiine ciuseu plumulel cumpiex is levelsibie Fuvmaliun uiine upen plumulel cumpiex isiasi anu illevelsibie anu niNinnniw W m yuiyesine eilmg uiine DNA assisieu bysigma V4 ineieinaiy plumulel cumpiex is a n asaawm iaimeuaiieiine isipnuspnuuiesiei band isiuimeuiz ni min ienglh inis Vvv Wi isine mus1 slabie plumulel cumpiex v anu is cumpiiseu ui enzyme DNA anu RNA Promoter recogn onIDNA binding i7 Cuie bindslu ianuum DNAWiin a naii iiie ui6E min inis is anaiameiizeu as iuuse binuing x ailhuugh ii isyeiy siabie Eiecliuslali mieiacliuns pieuuminaie 27 Huiuenzyme auuiiiun ui sigma snuws ai iedumiun inine leassucialiun cansianiiui binding ianuum DNA ine haiiriiie is lti sec Obviuusiy lhis is ieaiiy iuuse binding Whichiaciiilaleslhe seaicn iui a plumulel 37 Huwevei nuiuenzyme binds ciuseu plumulel DNAiEIEIEIx sliungei inan EDYE bindslu ianuum DNA Tne haiHiie is seyeiai nuuis Nuie inis is siiii pielly base but nui as iuuse as cane binding 47 Tne specmcilyuihuiuenzyme iui plumulel DNAys ianuum DNA isiDn bullheie isWiue yaiiaiiun inine amniiies ainuiuenzymeiui plumulel DNA Binding unsianisiangeiiuminuiuins Tne iaies uiiniiiaiiun iange iium isec iR NAgenes in i3D min iac leplessulplumulel Version 3 Transcriptional regulation Transition from initiation to elongation 1 Changes in conformation initially the polymerase covers the In bacterla region from 20 to 55 In early phases of initiation sometimes alter loss ofsigma the polymerase loses upstream binding and covers the region from 34 to 20 The general elongation complex forms alter 1520 bases have been transcribed and covers a region only 3040 nts Sometimes RNA polymerase pauses due to a temporary shortage ofthe complementary nucleotide When this occurs restarting synthesis requires the Gre and GreB proteinsto release the ause The 3 end is cleaved so that it is properly aligned within the catalytic site ofthe polymerase again 2 always left the promoter at the transition between initiation and elongation however it is now known that 70 of sigma remains associated with the core during elongation Which eukaryotic factor performs this 39 7 3 What isthe ratio of sigma to core and how much polymerase is same mum actually elongating TFiis A1 1 sigma3 core A2 25 Fin 9 21 Gene Transcriptional regulation How does RNA polymerase find in bacteria target promoters so rapidly on 9 1 Core RNA polymerase is P el 1Genera af nity muse binding VD a high intrinsic af nity for DNA which for DNA provides a larger target 395 39quotCreased by the Presence 0f than a promoter which is sci bp nascent RNA But its a inity for loose This results in a much faster rate kl b39quotd39quot9 539t95 5t h9h 0 allow the constant for association t a v a enzyme to distinguish promoters I IVIVI ef ciently 39om other sequences 2 random displacementquot between Mic5W tightly packed DNA instead of sliding r T along the DNA Zi fx soon WATS a I quot9 a 2 Fig 926 EDUrlEIEIEI cure HZWES a a rose uinpiae re enzyme and holoenzyme are distributed on DNA and very little RNA polymerase is free EDEHEIEIEI huluenzymes watluuse umplexes 2 By reducing the stability of the loose complexes sigma allows the process to occur much more rapidly and by sta quotzing the association at tight binding sites the factor drives the reaction irreversibly into the 7 nee huluenzyme formation of open complexes W What percentage is actually engaged ouuinnu 3 When sigma is released the core in transcription huluenz mes plus nascent transcript ternary grif n complex is essentially locked V r rumplexes at promoters onto the DNA until termination Du cure enzymes engaged in transcription Fig 914 Fig 922 Version 3 Transcriptional regulation in bacteria consequence of As a Underwuund Transcrlbln Overwuund transcription DNA In front of the eve supereells DNA 9 we supereells polymerase is overwound and DNA behind is underwound To compensate two enzymes are needed gyrase in front to Tupulsumerase Gyrase Introduce negative supercoils relaxes Ave in reduces eve s bereells su er ll andto I o p39som errase trailing t relax the supercoils Most ofthe supercoiling ofDNA in a cell probably results from F39Q 9 31 transcription Sequence Structure of the Core Promoter 4 1613 T82T84G78A65024A45 T80A95T45A60A50T96 Why is the distance between the 10 and 35 sites so conserved What types base changes would lead to increases up mutation or decreases down mutation in promoter activity 4 1613 T82T84G78A65CZ4A45 T80A95T45A60A50T96 Polymerase initially binds promoter at the 35 site 10 site is involved in melting the DNA strands Where would mutations affect the rate of form ation ofthe closed promoter complex Formation ofthe open prom oter com plex RNA polymerase overvvinds supercoiling the DNA in front and underwinds the DNA behind during elongation negative positive supercoiling supercoiling Topoisom erase the Gyrase precedes follows the polymerase polym erase introducing negative allowing positive supercoiling supercoiling RNA pol interactions with template Filter binding assay 57533 m Ff gP Protein 65 e 6o 6 0 IL 99 32Plabeled DNA G filter Nitrocellulose filter retains ProteinDNA complex RNA pol interactions with template Filter binding assay competition mewDNA complex 32Plabeled DNA NOTE pruteln eernes err er labeled N39quot quot39f39 se DNA and reblnds ente unlabeled lter retalns 3 UN A mm is trapped an the fllter but net detected complex 9U Version 3 RNA pol interactions with template Filter binding assay 39v anew t n Huluenzyme halfltfe eguals Effe uftemperature un the 3D to u huurs Curehalfltfe dtsassectarten ufpulymerase is less than 1 min huluenzyme 39nm DNA Conserved domains of slgma Means DNA Regictn 42 lnvuived in 735 recctgnitictn GSTcsigma 42 binding to prcrnctter in presence ctr umpetltur DNA Adlva 1 increase n rate but netelengatien r 2 Conla s wnh core llz eccur Ihroughoul its length 3 Can not bind DNA unless in undte cerelmust remove hiloly re on ti a 3 a Decreases an cereler nonspeci c DNA 5 Recognizes bases in nonlemplale strand at em and also double strands at e1 nrntnnndas RENE 23 insensintnriainrernntemi mama WWW tor nomads nnsmm 4 2 4 i 3 241 i i i l TTGACA TATAAT 735 4D 1 E9 2 372 A new recegnizes TAntTATAAT on quoton template strand Arnrnatic ammu acidstrpm and tyr43D make cnntact and prumuie melting Crystallizedreginn 23 netine inninitaw ul tltt l rrGACA YAYAAY 5 ct i M 6 Region 23 and 26 lorm continuous helix hinds e11and 712 and promotes melting u Irp and tyr residues hind DNA 725 region binds In the exlended e1 These promoters do not have a 35 reg on Represent 1 in E coli huI5 some m positive organ39sms n sigmaleaves tony 3nl alter my are transcribed signi es start oi elongation mailing 242 3 i 42 4t 3 3 251 TGnTATAAT 35 71D 1 Extended 40 prom oter Version 3 9 Reg39 n 11 is Ihnughlln maskreginn 52 mm 39nding DNA u a inm between cure and reginn11disrupllhe mas u nlregin 52 1n Prunuseutnat entry ut RNA intutne ex39t cnan n RNA nui cure is the aiiusteric etiecturtnat uissuciates slgmz 11 A ivmnls cuntact eithertne alphzsuhunll ut cure nrsigmz intneu re 39nnTwn rnuues nla 39 39nn ent ut RNA nui nnua nrurnuter lads 35 but has sitetur PhnB nrutein mlher nus ht enhancing subsequent step In Irzmcnpllnn tiess clean 1 renus nning nuiuenzyrne CAP renusitiuns recngn un nei39 ixatas H 42 M a uunmasked in binding cure CTGGnA TTGCA RNA pol CORE Nitiugen starvatiun an CTGGnA TTGCA Interaction ofRNA polymerase with template me am Wm holoen 1Vquot e W We uuww um mm a We Transcriptional regulation in bacteria Interaction ofRNA polymerase with template Sig a leaves attei 1145 nt ave uansenbed Is the lt15 region always located at35 nucleotides upstream from the start of transcription Gene tactur use 35sequence senar un e1n sequence rpoD d39 eneiai TTGAC 1313 an TATAAT an heatshuek CCCTTGAA 1315 an CCCGATNT VpoE of heatshuek nutknuwn nutknuwn nutknuwn aquot mtngn CTGGNA Ebp TTGCA if agella CTAA 15 an GCCGATAA Fig 9 35 Molecular Genetics PCB4522 Spring 2004 Lecture 2 Replication Dr Eva Czarnecka Verner Course web page http PCB4522IFASUFL EDU Dr 90 to Microbiology 8 Cell Science home page and look under course In aterlal Chapter 13 DNA Replication Chapter 15 in Gene VI amp14 in VIII Primosome a protein complex that initiates synthesis of a DNA strand Replisome complex of proteins engaged in elongation of the newly synthesized DNA strand Assembles at the replication fork Identi cation of protein components involved in DNA synthesis 1 temperature sensitive mutants conditional lethal mutants replication at permissive conditions but fail to function at nonpermissive conditions high temp 42 C In E 601139 identi ed loci dna genes 2 dna genes a quickstop mutanw immediate stop in replication elongation enzymes defective amp defects in precursors b slowstop mutants defective in reinitiation smaller class Identi cation of protein components involved in DNA synthesis 3 in vitro complementation systems combine extracts from mutant and wildtype strains Can add back purified proteins to identify function ofa specific dna gene product Progress much slower in eukaryotes DNA polymerases enzymes that make DNA 1 Both bacteria and eukaryotes contain multiple DNA polymerases 2 The ones that actually replicate the DNA are called DNA replicases 3 All have the same type of synthetic activity a each can extend a DNA chain by adding nucleotides one at atime to a 3 OH end b the choice of dNTPs dictated by base pairing with the template strand DNA polymerases enzymes that make DNA 4 Some function as independent enzymes 5 Bacterial DNA replicases contain a large number of subunits large protein assemblies It is hard to say which proteins are actually subunim and which proteins are just loosely associated Five DNA polymerases in E coli Q 1 poll encoded by polA gene a Major m enzyme for damaged DNA b Plays secondary role in semiconserVatiVe replication c Most abundant 400cell d Molecular mass of 103 kD G 2 pol H encoded by polB gene a Minor DNA repair enzyme b Molecular mass of90 kDa Five DNA polymerases in E coli 3 pol III encoded bypolCde gene a REPLICAS E39 de novo synthesis of new strands of DNA b Contains many subunits c There are 1020cell c Molecular mass of900 kDa c Has no 5 to 3 exonuclease activity Five DNA polymerases in E coli 4 pol IV encoded by dinB gene a SOS repair enzyme ofdamaged DNA 5 pol V encoded by umuD ZC gene a SOS repair enzyme ofdamaged DNA Phage coded DNA polymerases 1 T4 T5 T7 amp SPOl 2 Mutations prevent phage development 3 Each phage polymerase peptide associates with other proteins phage or host to make the intact a 5 3 synthetic activities b 3 5 exonuclease proofreading activities enzyme Eukaryotic DNA polymerases Five identified in mammals Enzyme all 50 5m B I Location Nuclear Nuclear Nuclear Nuclear Mitochondrial Jnction priming elongation repairamp repair replication fboth ofboth replicati strands strands 355 No Yes Yes No Yes exonuc relative 80 1015 215 activity PRIMASE REPLICASE Eukaryotic DNA polymerases PRIMASE DNA Pol aI39primase complex bifunctional 48 kDa PRIMASE initi complementary RNA prim Heterotetrameric phosphoprotein ates DNA synthesis makes er 58 kDa protein tethers primase to 180 kDa subunit 180 kDa polymerase A subunit extends RNA primer by making a proofreading activity 70 kDa subunit no known catalytic function may recruit polo primase to the replication fork short DNA only in Drosaphila has Eukaryotic DNA polymerases REPLICASE DNA polymerases 5 III and 5 II 1 Heterodimeric 2 Need auxillary proteins a RFC replication factor C binds to RNADNA primer amp stimulates assembly ofpol 5 or pol s b RFC loads PCNA processivi factor onto DNA PCNA binds to 5 or s amp makes them stable on DNA 3 Intrinsic proofreading activities 4 Probably synthesize all cellular DNA 5 t0 3 DNA synthesis occurs by adding dNTPs to the 3 end of the strand 5 PPP 3 OH PP Choice of dNTPs GC ArT 6 DNA synthesis has an extraordinary high delity between 398 and Q40 1 error per genome 4200 kb per 1000 bacterial replications Substitutions frame 5 39 7 Proofreading function all bacterial DNA polymerases have a 3 5 exonuclease activity Operates in the reverse direction from synthesis Processivity 8 In proofreading the excised base is replaced by a different active site of the enzyme than the one used for the original synthesis Expected error is1 per 1000 bps replicated Proofreading drastically reduces the errors made in replication Enzyme Synthetic Proofreading Err r dom 39 omain proof proof DNA polI aa 200600 Nterminal 10395 5 x10397 DNA pol 111 o subunit 2 subunit 7 x10396 5 x10399 T4 DNA pol ctenninal Nterminal 5 x10395 10397 Rev tmnscrip none 10395 E 001139 DNA polI poll easily cleaved into two fragments by proteinase a large fragment Klenow fragment contains polymerase and 3 5 exonuclease proofreading domain Used in vitro for synthesis reactions DNA sequencing Klenow fragment 68 kD small gment 35 kD N IRVVllllll C C xonuclease polymerase 3 5 site proofreading catalytic 5 3 exonuclease in vitro DNA synthesis using the Klenow fragment DNA pol 1 Experimental Uses a Fillin reaction to label recessed ends of DNA EMsA N soutnem b DNA sequencing Unique ability to stan replication at anick in DNA Klenow fragment DNA pol I Catalytic domain of T7 DNA Polymerase Large 5133 Right hand sunmm synthetic I lmnain has 3 parts DNA pol 1 pall small fragment has 5 to 3 exonuclease only This activity allows pol I intact to be used for nick translation m v BkDa Klenuw agment I KSkDa small fragment I I exunuclease pulymerase 35539 Q exunuclease 55339 qu 39eadmg aumain Nate remuves RNA pnmer amp1prDN DNA pol I poll 2 Nick translation initiates at nicks in DNA Extends the 3 OH end while removing the strand in front by its 5 3 exonuclease activity Displaces existing strand 3 DNA pol 1 plus DNase is used for in vitro labeling of DNA by nick translation Nick translation by intact DNA poll 1 add DNase to nick DNA 3 OH 5 P 3 5 excised DNA fragments x 2 gtxlt 1 do 93 gtxlt P 5 5 3 Add one ZIPdNTP three cold dNTPs and DNA poll note rLick moves 5 to 3 E coli DNA poll in viva function Filling in short stretches of singlestranded DNA that arise from a DNA replication lag g ing strand b DNA repair when damaged bases have been remove E coli DNA pol III Replicase 1 In order to study must usepolA mutant strain ofE 601139 since the pol I concentration is so great that it verWhelms pol III activity In vitro studies use extracts from polA mutant cells 2 Subunit structure Q a 0L subunit 130 kDa DNA synthetic activity dnaE Mutation is lethal Q b a subunit 3 to 5 exonucleolytic activity proofreading function dnaQ Mutations increase error rate by 103 Initiation of DNA synthesis All DNA polymerases require a primer to provide a free 3 OH end to initiate DNA synthesis Types of priming reactions 1 Primase synthesizes a short RNA primer that is then extended by DNA polymerase cellular DNA papova virus 2 Extension ofthe 3 end ofDNA at anick rolling circle replication oflt1gtX174 3 ProteindNTP primes directly by presenting first dNTP adenovirus bacteriophage 4 Preexisting cellular RNA mitochondrial genome retrovirus ColE replicon requires along RNA primer ng 12 34 1236 1237 Genes v11 1 Transcriptionr 555 bp RNA primer upstream from origin of replication passes origin has three hairpins 2 RNase H cleaves RNA primer at originr free 3 OH 3 Persistent 7265 720 RNArDNA hybrid remains 4 DNA synthesis starts replication 5 Control a RNA primer precursor is a positive regulator b antisense RNA I 108 b is a negative regulator c 39 A IRNA primer binding Rom lprotein e N what in ibits replicationr transcription continues 6 Mumu39ons in RNA 1 and RNA primer pairing region 39 quotquot 39 quot giuun DNA synthesis is semidiscontinuous and primed by RNA The problem DNA synthesis must always proceed from 5 to 3 As the replication fork moves one of the template strands continuoule ex oses new u stream tem a e Egt 3 5 lagging strand leading strand 3 5 Semidiscontinuous replication leading strand synthesis can proceed continuoule i the 5 to 3 direction lagging strand synthesized in the reverse direction as a series of fragments which are later joined Discontinuous s thesis m 3 5 lagging strand leading strand 5 3s Semidiscontinuous replication The lagging strand fragments are known as Okazaki fragmenm Usually 1000 to 2000 bases in length 3 lagging strand 1 RNA primer 1112 bases RNA polymeras FdnaG primase Semidiscontinuous replication The leading strand is also often isolated in fragments due to the misincorporation of UTP Repair of the UTP leaves small gaps until they are filled in pseudoOkazaki fragments UTP leadingstrand gap 5 nununruuuununnun 3 Semidiscontinuous replication Steps in lagging strand synthesis a synthesis of RNA primer b extension of Okazaki fragment DNA 0 synthesis of next Okazaki fragment upstream of the last d removal of RNA primer Who s done it e fill gap and seal ligation nick Semidiscontinuous replication DNA pol I polA starts synthesis at the nick between DNA and RNA and 5 3 exonuclease activity removes the RNA primer and replaces it With DNA DNA pol I lagging stmd 2 nick r RNA primer 1 5 3 nick translation 5 3 DNA ligase l LigaseATP complex formed 2 The ATP changes to AMP as is releases pyrophosphate PR and covalently attaches to the phosphate of the 5 end of the DNA 3 Original phosphate at the 5 end covalently joins the OH of the 3 end and AMP is released NOTE T4 phagehgzseusesATF E 5011 hgzseusesNAD Ligation O DNAligase fpolNADorATP Q o T4phagengaseusesATp 030 E colt ligase uses NAD 5 Ligase seals nicks in DNA Tie eanfkcture 2 There are two types ofDNA repueatror in E Call 1 c1gtX174 phage 2 OriC origin otbactenal cnrornosornal repllcatlon There are two types ofDNA repueatior in E can 1 lt3wa pnage eaen strand Syntheslzed separately unldlrectlonal repllcallon fork m A Synthesls ottne e strand to torrn tne doublerslranded serves as a model for agglng stran synthesls b Synthesls ottne strand to torrn Slnglerslrands for packaglng lnto pnage pamcles servers as a model forleadlng strand sysnlhesls 27 070 orlgln of baclenal enrornosornal repllcallon botn st rands syntheslzed at tne sarne trne bldlrecllonal repllcallon fork DX174 phage as a simple model for replication Rolling magma strsle swan Wis Wm gt O O gt O gt gt O strand Replicative form RF ds plasmid strarrd packaged to form virion R01 gc clerepl at n amode1 for 1eadrrrg strarrd synthesrs mm phage replication provides a model forDNA replication R F mummymcked sea 0 4 Rep hEllcasE DD Egt Egt g W Eackground for Rolllng circle repllcatlon Note no DNA polymerase JUSLLO separate Single strands Two kinds of activities are needed to conven double stranded R DNA to singlestranded DNA without N synthesis ofnew a Helicase separates the strands using ATP to provide the energy b singlestrandbinding protein SSB CIDX174 as a simple model for replication Rolling circle replication REFA 0 SEE Proteins needed for rolling eirole replieat e Aquot r n ion u ntoniekatorigi pas CovalentlyllnkedtoS end ofthe displaced d an b SSB protein to keep DNA singlerstmnded Binding is highly coop erativ e c Rep protein provides heliease function d DNA pol III holoenzyrne Molecular Genetics PCB4522 Spring 2007 Lecture 1 The Replicon chapter 13 Course Web page httpifasu edu or no In Micrnmnunny a Cell sciincc home my um lnnk undercm se Morin Chapter 13 THE REPL IC ON Replicon aunit ofDNA in which individual acts ofrephcaiion occur contains an origin replicaiion start inus occurs once for every cell division examples bacterial chromosome plasmid mitochondrial genorne r Virus Bacteriaquot plasmids amp Phage have single replicons ONE On ufREFLlCATlON a i Bacteriophag swam W M Plasmid autonomous circular DNA genome that constitutes a separate replicon single copy control replicates once alongside bacterial chromosome 7 multicopy control present in more copies than bacterial chromosome How many origins of replication in a plant cell g Ro Nucleus chloroplasts Mitochondria General rules for replication 1 Initiation ofDNA replication comm39 39 39 39on st not occur until after replication is complete Note Replication is controlled at the mga nf39 Types of replication control 1 Single copy bacterial chromosome some plasmids eukaryotic chromosomes note Eukaryotic chromosomes have my replicons39 control is a problem all must activate w in cell cycle but no more 2 Multicopy some plasmids mitochondrial and chloroplast DNA l Replicons can be linear or circular H Origins can be mapped by electron microscopy autoradiography amp gel electrophoresis l replication fork point at which replication is occurring growing point 2 may be unidirectional O 1C replication eye 15 3 or bidirectional Ori u 1 7 bacterial and eukaryotic C s chrom o som e Replication eye forms a theta structure in circular DNA two daughter duplexes replicating 8 structure Appears under EM 1 as 9 struc ure ColEl plasmid DNA from E Enli ls replication unidir 39 or bidirectional runly ne and muves umdlrechunal Drnsnph ilz DNArepliczlinn studied us39 a dmzrenlizl labeling DNA replication studied using zn gel electrophoresis 1n amenml by mm 2quot directinn by SM1 2 sum ulmrmiun pulse gel Elihum f electruphnresis mu mm n i an mm o 4 Pk l MW Bacterial chromosome is a single replicon circular bacterial origin supports a initiation of replication b controls frequency c segregates replicated chromosomes Usually associated with the origin 1M in euka otes Bacterial chromosome is a single replicon 1 Replication normall terminates when replication forks meet 2 Tor sites serve as back to prevent replication lrom going too lar 3 Transcription olmajor genes is in the same direction as replication Replicau39o lica on 2 1 tor sites 23 hp and function in Replication termination and only one orienla 39on gene O emztion in bacte a 2 Protein from the mr gene binds to m site and blocks replication lork 3 Replication iorh moves circa 10x raster than RNA polymerase a Lfboth moving the same direction then DNA pol must slow down or run through abort tmnsx b opposite directionsdif cult Therefore mos major genes have the w orientation as replication Identi cation and isolation of uriC ari if 71 1 clone random DNA MES cunmmng amp renmnce cmmining media m salect rq imte have m39iC 3 cuttheinsma ll Aintn er 1 r reclnnetnidentify the smallest piece am still can Amp resimnt vector whim Identi cation and isolation of uriC Smallest fragnent am can mppm replication 2 segegztinn function pm lnmterl elsewhere 3 very Ammh mm my neededtn melnhe DNA Identi cation and isolation of uriC 01139 C meme WY 3mm utensil also serves as aprotzm hndnngatzDmA Each eukaryotic chromosome contains many replicons L Hard to measure size nfzva39zgerzplicnn since zdjzcmt ones rose Thought to be from 40 to ma kh 2 Nn termination signals Terminate by meeting one in 39 1 mx slower in ulkzryntes 3 Active replicoris are clustered sunesun active at any giver time 4 ll ali replicons active replication conid be complete in 1 hr h t s 39 39 nase lasts aoontohr Tinsnnpliestiiat only 2h out 15 are active at any giver time Eukaryou39c nucleus contains from 100 to 300 foci One rocns may cnnmin gt3lm replicons Cells labeled with lirdu to identity replicating DN L el39t panel stained using zn rmd antibody Right panel shined with propidinrn iodide to see all DNA Eukaryotlc Initiation of replication in three steps DNA Origin recognition proteins oRPs repllcatmquot bind to origin orreplication 0n ikfmai m 0 9 quot oARs scantymooomooslyrepliconng sequmce when pom foreign DNA rephcnlm begns demon sites nudeupmlem cump ex n o1 o llellcoseoctwit lrloereotwonnolsps direcl39 nal y syn lmsIs o axORPsdistmDNA bulmm olso recruit v1th protemsto complete to Jab E39o Eukaryotic DNA replication 3 Generally bidirectional Origin 4 Genomes replicate once per cell division eukaryouc ce Excepnuns a endoreduplicau39on gene ampli cation cell speci c during animal development Replication racto b mitochondrial multiple times nucleoprotein throughout cell cycle synthesis Eukaryotic replicons are small and replicate more slowly than bacterial DNA Organism Replicons Length Movement Bacterium 1 4200 kb 50000 bpmin Yeast 500 40 kb 3600 bpmin Fruit fly 3500 40 kb 2600 bpmin Toad 15000 200 kb 500 bpmin Mouse 25000 150 kb 2200 bpmin Plant 35000 300 kb 7 Mitochondrial origins or replication i Purple sulphur bacteria able to handle oxygen ancient symbiont ancestorto eukaryotic mitochondria 2 Mitochondria are usually transmitted by the female 3 We all have maternal mitochondria 4 How is mitochondrial dsDNA replicated a use different on to initiate replication of eacn DNA Strand b replication oanstrand starts nrst amp in a D ioop new L strand produced 0 replication of testrand starts new Hnstrand produced wnen on on tne oid L strand i5 exposed bythe replication fork movement RNA pnrnern tnen DNA Mitochondrial DNA D loops Replication ofmammalian mitochondrial Orlgln un 0mm H strand DNA has segarate origins foreach strand L strand 39 L stran D p dlsplaced L strand l5 Slngle stranded Synthesls ofnew L Strand L Note onng forL strand l5 on the H strand 1 RNA pnmersymhemed by RNA The snort strand l5 unstable and polymerase L mud d placed turns over 500600 bases 3 end cutb a 37mm speci c mdonuclese at Specific sites 3 OH extended into DNA by DNA polymerase N 1 G VIII Replication of mammalian mitochondrial DNA has separate origins for each stran QB 62 n w Orlng for H strand lS un tne L strand 1 Shun RNA pnmer synthesized by pnmase SVWES S Di quotW H St gm 2 339 OH extendedmtu DNA by polymerase relatlye D New L stran Partially replicated amp Duplex circle lt3 G Q Dup 16x L L 39 circle Gaps ll l new strands are sealed Mitochondrial DNA D loops 1 Single D loop displacement loop opening of 500600 bp in mammalian mitochondria 2 Tetrahymena mitochondrial DNA has 6 D loops at a time many origins and plant chloroplasts have 2 T ew short strand is unstable degraded and re synthesized to keep the loop open 3 Starts by synthesis of short RNA primerwhich may be extended by DNA pol this is normal for all DNA synthesis 1 G VIII Mitochondrial DNA D loops The displaced strand of the Dloop remains single stranded until the origin on the L strand is reached This second origin initiates H strand synthesis This illustrates the principle that an origin may be used to initiate a single strand only in the case of D loops and rolling circle PhiX174 modes of replication Rede ned angm a regtlcanan An origin can be a sequence of DNA that initiates DNA synthesis using one strand as a templa e The problem of linear replications Since the new strand is always synthesized in the 5 to 3 direction it is easy to nish the new strand by running off the ofthe template but how does the polymerase initiate at the 3 end ofthe template strand DNA pol usually binds region g that surrounds on In F g 1112 a VI 5 The problem of linear replications Solutions 1 convert linear to circular or multimeric molecules rolling circles 9 a 50 l examples lambda 7 phage is circular and T4 phage multimenc The problem of linear replications Solutions 2 create unusual structure at the end example haiggin so there is no free end or linear mitochondrial DNA of Paramecium crosslinks the ends 3 Q 5 The problem of linear replications Solutions 3 end may be variable example Eukaryotic chromosomes have short sequence repeats at the termini telomeres A separate mechanism adds or removes these repeats It is not necessary to replicate to the end as long as some of the repeaw are copied The problem of linear replications S olutions 4 protein intervenes O en used by viral nucleic acids that have proteins that are covalently linked to the 5 terminal base adenovirus DNA 80 kDa phage 129 DNA poliovirus RNA 22 amino acids Strand displacement Linear template Adenovims 1 bottom strand is used as template and the top strand is displaced 2 top strand forms terminal duplex before initiating DNA synthesis Free single strand 5 5 3 i 5 j 3 3 3 3 5 5 3 Dupiar ongin formed 3 3x 5 by base pmng Re licated indg mdentlz Strand displacement Linear template Adenovirus 1 Terminal proteindCTPDNA polymerase complex binds to 5 end ofthe top strand ofadenovirus DNA 2 3 OH of dCTP serves as aprimer for DNA synthesis note deoxyribonucleotide 3 New strand is covalently linked to the initiating dCTP 4 Old TP is displaced by the neWTP for eaeh new replication cycle 3 Strand displacement Linear template Adenovirus 1 Terminal proteindCTP binds to 5 end ofthe top strand of adenovirus DNA between 9 amp l8 nucleotide 2 Host protein nuclear factor I essential for the initiation binds between 17 8c 48 nucleotide 3 Initiation complex forms between positions 9 and 48 at a xed distance from the actual DNA end NH 5 DX174 phage as a simple model for replication rolling circle Rolllng strand Replicative form RF ds plasmid strand packaged to form virion Roll cle rep cat on amodel for leading strand synthesis Molecular Genetics PCB4522 Spring 2004 Lecture SReplicationpart D Dr Eva Czarnecka Verner Course web page httpPCB4522IFASUFLEDU or go to Microbiology 8 Cell Science home page and look under course in aterlal Replication requires DNA Polymerase III In E mli 1 Single type or catalytic subunit dnaE used in replication orboth strands 2 Active replicase is a dimer each hair enzyme unit contains dnar I S subunit amp o er protein In B mbtilix 1 Two dirrerent catalytic subunits a Pol J homolog orE colidnaE synthesizes theleading strand b dnaEBS synthesizes the lagging strand In eukaryotes 1 The same overall structure oI DNA pol 111 2 nquot 3 Not clear whether the same or different catalytic subunits used Replication of E coli chromosome Genes VIII Chapter 14 Replication requires DNA Polymerase Ill 1 DNA polymerase III holoenzyme 900 kDa complex a a catalytic core g subunit dnaE b a 3 5 proofreading 5 subunit dnaQ c a Q subunit that stimulates exonuclease d a dimerization component 1 that links two cores e a processivity component I that keeps polymerase on DNA clamp f a clamp loader 1 that places the processivity subunit 1 on DNA complex of 5 proteins Forms of DNA polymerase 111 from biochemical studies 25 kD proofreading El gt10 kD 130 kD structural holds tallquotC together increased processivity Egt 71 kD 1 maintains dimen39c structure Forms of DNA polymerase 111 from biochemical studies 15 complex makes is clamp bind to primed template Asymmetric Clamp loader only one V5 mplex com lex Ir ofli 39y adds a pa dimers Assembly stages for P01 111 holomzyme 15 is the clamp loader 5 dimer 87a recognize primertern plate 6w 0 3 60 Tilt dimer clamps on DNA increased processivity Corejoins complex x form s stru cm r Assembly stages for P01 11 holoenzyme 2 9 Wkan suhmtsazeencadedbylhe meDNA 7 Agmmecric nnly mm clamp linden why Lagging strand Leading strand Aleferent dim eric almimes D e dissuclate noloenzyme 5 subunits of DNA pol 111 head to tail dimer 12 pinchesstall symmn39y mamquotmm DNAv39n watt ninkcuks The clamp 3 Ful ill 5 Replication of E cali chromosome EgtdnaB helicase Replication of E cali chromosome Replication of E coli chromosome Replication of E coli chromosome RNA p rim er Replication of E coli chromosome The template for a lagging strand is pulled through creating aloop in DNA Replication of E coli chromosome O The template for a lagging DnaG 5 strand is pulled through creating a loop in DNA Replication of E coli chromosome 2111 Core Pol III The loop is released w 7 1st CorePol 111 Replication of E coli chromosome New clamp 8 Core P01 111 present on DNA Organization of the oriC Replication Fork ATP ham Dual DnaG primase stimulated by DnaB Note theloading oI DnaB helicase by DnaC only occurs atthc origin 3 5 fragment What is responsible for recognizing the sites for initiating synthesis of Okazaki fragments Dual properties of dnaB helicase 1 Propels the replication fork 2 Interacts with dnaG primase at a correct Semidiscontinuous replication The lagging strand fragments are known as Okazaki fragments Usually 1000 to 2000 bases in length lagging strand 1 2 site RNA primer N1 112 bases RNA polymerasednaG primase On C Primosome Schematic of directed synthesis meld Of le rephcation fork DnaB Role of DnaB Related 1 propels the DnaG fact replication fork primase At aiiC the through its stimulated pnmosgme helicase activity by DnaB consists of 2 required to naB a activate primase DnaG DnaG OriC Primosome 1 binds dnaB to attach pol directed Synthesis 111 core to replication fork 1 Speed of Role of Pol 111 DNA 1 Synthesis of leading syn39hesis strand Increased 10x 2 synthesis of lagging ZICZV md strand by extending the fmmgfamng RNA primer Displaces offincrcascd primase roccssivity 3 pulls the lagging strand template through the an G holoenzyme anase H mp mum is m 4 e u m mlymmMIl Can nedar Joins mime In two POW Inltiauon silo current Dr nexl anznkt 0mm I d mm fragment um um Irglta m Euka otic DNA pol mprimase DNA Pol al primase complex bifunctional Heterotetrameric phosphoprotein 48 kDa PRIMAS E initiates DNA synthesis makes complementary RNA primer of new strands 58 kDa protein tethers primase to 180 kDa subunit 180 kDa polymerase A subunit extend s RNA primer by making a short iDNA 2030 bases only In Drasaphlla has proofreading activity not V processlve polymerase 70 kDa subunit no known catalytic function may recruit polot pnrnase to the replication fork 000 Eukaryotic DNA Replication 1 DNA pol LQprimase a initiates synthesis oilagging and leading strands b RNA 10 bHDNA N21173 b primer 2 DNA pol sa a elongates leading strand continuously b highly processive interacts with RRC amp PCNA c can dimerize w also elongate the lagging strand 3 DNA pol a a may be involved in lagging strand synthesis b other iunctions 4 Replication iactor 1 RFC a clamp loader binds to 339 end oI iDNA amp loads PCNA b ATPase activity used to op en PCNA ring Eukaryotic DNA Replication 5 PCNA proliierating cell nuclear antigen a tethers DNA pol d to the template b acts as processivity iactor ior strand like 9 clamp elongation d trimer iorms a ring that surrounds DNA 6 Replication iactor RFrA a single strand binding protein Topoisomerases 1 amp 11 maintains DNA winding 3 ExonucleaseMF1 a removes RNA primers r 9 T antigen helicase T antigen loadinghelicase 9 DNA ligaser Eukaryotic DNA Replication Nutes Euka otic replication fork contains one complex of DNA polotprimase amp two other pol complexes either 2x 5 s or one 5 amp one 52 In mammalian systems DNA pol has no 5 3 exo activity Okazaki fmgments removed by a RNAse HI speci c for RNADNA hybrid endonuclease cuts b FENl exonuclease removes the RNA 5 3 Similar functions at bacterial and mammalian replication forks Function E coli HeLaISVAO helicase DnaB T antigen 1oading helicase Dnac T antigen single strand SSB RFA priming DnaG Pol otlprirnase sliding clamp p PCNA clamp loading ya RFC catalysis P01 111 core P016011 holoenzyme I m dimen39zation RNA removal PolI MFl exonuclease ligation ligase ligase 1 Origins l ColEl RNA ll acm as primer 2 CDXl 74 replicative form 3 oriC CulEl plasmid RNA ll primosome wequot Creating replication forks at oriC l the strands melt at the origin over a short distance 2 DNA is unwound 3 first nucleotides synthesized into RNA primer Occurs only once for leading strandmany times for lagging strand Creating replication forks at oriC l Initiation at oriC starts with complex formation of 6 proteins a DnaA DnaB DnaC HU Gyrase and 2 DnaA uniquely involved in initiation 3 DnaBC engine of initiation at origin Minimal oriC O DnaAbindigg Region ofmelLing I E I N w a l 37m er s l 9rmers 245 bp GATCTNTTNTTTT TTATNCANA Note GATC is Dam melhylau39on site 11 copies oI GATC in oriC Creating replication forks at oriC 1 Binding of DnaA to four 9 bp sites at on right side of the origin 2 24 DnaA monomers form a tetramer and DNA melm at the three 13 bp sites on the left side 3 DnaBDnaC joins the complex to form bidirectional replication forks Minimal oriC 274 monomers bind cooperatively DnaA protein gt 4 9rmer sites DNA strands melted at 3 137mer sites DnaBDnaC VDnaB hexamers gt 2 DnaB binds amp displaces DnaA from 13 bp repeats Minimal oriC Q Does DnaA act as the titrator that measures number of origins vs cell mass A Mutations DnaAreplication asynchronous overproduction of DnaA39 39tiation starts at reduced cell mass ATP AMF lt1 DnaBDnaC gt DnaB hexam ers Creating replication forks at an origin Other proteins are involved in replication 1 Gymse acts as a swivel allowing one strand to rotate around the other 2 SSB stabilizes single stranded DNA as it forms 3 HU HUIHUZ general DNA double stranded amp single stranded binding protein Bends DNA structural role Similar to histones M cooperativity in binding Causes DNA to bend and fold into structure that leads to open complex formation amp resembles beaded chromatin Creating replication forks at an origin ATP required in replication 1 For helicase to unwind the strands 2 For gyrase to swivel strands 3 For primase to initiate 4 For DNA pol III to be activated Methylation state of DNA may regulate replication Active 1quot origin GATC 13 miquot Single round CTAG delay N BlIe replica on a Dam methylase GATC CTAG Me Active origin methylated DNA methylated DNA Me hemimethylated DNA accumulates Methylation state of DNA may regulate replication Membranebuund inhibier competes with DnaA for nriC V e GATCnn nnnnnnnn h th I t dDNA CTAGnnnnnnnnnn am y Inactive origin Dam methylase delayed 13 min at oriC vs lt1 5 min for GATC elsewhere in the genome Me DnaA protein I Inhibitor released DnaA can initiate Methylation state of DNA may regulate replication Squ inhibitor binds to hemimethylated DNA what delays rereplication N Squ may interact with DnaA Equot emimethylated origins bind to cell membrane inaccessible to methylases 4 Methylated oring do not bind to membranes 5 No clear connection between the origin and membrane 3239s end of fecture 5 Molecular Genetics PCB4522 Spring 2007 Lecture 4 chapters 13 and 14 Control of Plasmid Copy Number The eukaryotic ORC Control of Plasmid Replication Plasmid incompatibility is connected to the regulation of copy number and segregation l Compatibility group aset ofplasmids that are unable to co 39 39 ebacterialcell a Both plasmids have the same type ororigm b For the ColE1 plasmid copy number is controlled repressor RNA 1 that measures the concentration of origins 2 ColE1 plasmid employs negative control for its copy ber and incompatibility system ca 20 copies per cell Sel sh plasmids with territorial rights Plasmid incompatibility is connected w39 copy number control 1 Primer RNA 555 bases starts upstream of the ori and extends into the ori a cleaved by RNase H cuts RNADNA hybrids b 3 OH of RNA serves as primer to initiate DNA synthesis 0 only cut by RNase H if it is not duplexed with antisense RNA RNA 1 Replication of ColE1 plasmid DNAmulticopy control Replication starts with transcription RNA II is required for m DNA synthesiS RNA polymerase 39t39ve gu at on 555 3920 Pl and Pll are promoters forthe regulatory transcripts RNA I is complementary to the 5 terminal region ofRNA primer II Replication of ColE1 plasmid DNAmulticopy control RNAJoop structure 39 d 0 ds stem ss loop Replication of ColE1 plasmid DNA multicopy control 265 20 replication Replicase Replication of ColE1 plasmid DNA mu ticopy control 555 265 20 pgt origin Pl RNA 39 RNA ll disrupts the RNA loops Replication of ColE1 plasmid DNA mu ticopy control 555 20 P39IIEgt origin No replication but transcription ontjnue thhout loops RNAII is notable to form a persistent hybrid rigin Plasmid incompatibility is connected with copy number 1 RNA I antisense inhibitory transcript forms duplex with Primer RNA II 2 The Primer RNARNA I duplex molecule is not cut at ori by RNase H and the persistent hybrid at the origin is not formed 0 DNA synthes1s 15 not 1nit1ated g New incompatibility groups generated by mutations 4 Mutations in Primer RNARNA I interaction region ti may result 1n formation of a new compa blhg group RNARNA duplex can not be formed between the mutated RNA primer and the original RNA 1 The mutated and original replicons no longer regulate each other Original amp mutant plasmids behave as members of different compatibility groups Plasmid incompatibility is connected with copy number Q How does RNA I negative regulator counts the copy number A PI promoter regulates the expression level of RNA I regulatory transcript 1 At low levels of RNA 1 replication occurs 2 At higher levels of RNA 1 replication shut down Q How many levels of regulation A Positive Primer RNA 11 enhances replication by providing 3 OH end B Negative RNA I rgpressor shuts down replication but allows continued transcription of RNA 11 C Negative Rom protein helps to shut down replication because it enhances RNA primerRNA I duplex formation X t t t d k t39 Licensing factor controls eukaryotic replication e wpus eggsg z sl azlfnu y 9quot aryo 398 1 The eukaryotic genome tens orthousands orreplicons Each origin activated only once in a single cell division nucleus Remove nucleus One round of 2 A rate limiting iactor involved Used up by the rephcation D event at each ori Must be renewed alter cell division to allow iurther replication at that origin S quot round of synthesis 3 Egerimental System Xenupu eggsa can replicate DNA in a Block protein nucleus injected into an egg devoid or original nucleus Neg ptrutem wrest nthesis 39 39 39 39 ea 5 D No further DNA wi t any new gene expression Material with NA can armamean Bf vmhesis nuclear membrane bein the rorm ora sperm nucleus or an interphase nucleus Licensing factorrnust enter nucleus but can not pass the nuclearrnernbrane Origins of yeast replicons ARS Licensing factor controls eukaryotic replication autonomously replicating sequence One DNA replication licensing factor Egt 1 about 50 bp In length and very A inactivated o F 0 2 contains two domains Cell division a A domain 14 bp 1 bp core ARS consensus Critical for ARS function r t quotMlearmembm e Bin ing te forthe origin recognition complex us ORCwhich contains 6 proteins lt39 0 b B Domain mutations reduce orifunction Cylup rn 1 several B elementsimperfect copies of the new licensing factor L f t that 1 enters the nude equots quotg 3 0r 5quot quot core ARS consensus 911 single round ofDNA replication 2 transcription factor binding sites ccurs The yeast ARS The ORC origin magma The yeast ARS complex Origin recognition complex 1 6 proteins 400000 Dalton 400kDa 2 must bind ATP herore it can bind to the ARs core consensus Transcription lactor ABFl only enhances initiation B1 A domain core consensus Ongln can function A domain I l core consensus effectwely Wl l functional A ORC binds to A and BI domain and any imperiect consensus two B elements domains Events at A and B1 critical for initiation F 1441 c VIII 0 one haund In omhrnueh eel cycle Earn G1 Arm nmlerled again nlase hyrevsmsniue sne In an The yeast ARS CM cdessymheszed In 61 highly Cd uanme n1lt5 rrIn Warsquot Merri cnmplex reeruned In re Late G1 cdes G ARS hy cdes in mange cm mirgicaim werequot meme e eii cmviex snimsis Merv Drmans siesiwm inihe used m on y bound ddiing ei Mcm27 is licensing factor MW in nif madiiginaime J ORCmusATP v mid 13K ii wig M cmZJ is mmemmj Phasphnvvialed maintain binding unto the urigiri ORQ Cdeaic r and Memm can 3 be dewnreguiared V 0 51 V 7 aiihe 1i N minded er in Sphase DNAsanhesIs In med cdesaMem 26 New P W Mimi rumquots dsv39md Na and gems cinnka Cdc mnmly degraded m vnev g av quotg nn lunlllallnn 62 88 Mcmzq is licequot Licensing factor consists ni MCM proteins Genes vmiciiageru 39 The ORC u MWES ATP m ORC hydxnlyxes ATP and lnarls Memu grim DNA as K e dertu iead m 2 E39PY IIE I ng i 7 Mcm en piex 047 Q in v ORC identi es are irrigan nfreplicatinn rnr Cdn Call amp MCM Only licensed nrigjns enmaining McmL7 can replicate The helimse activity nf Mum unwindsthe DNA ahead nf are replicatinn fm39k and mnves wmr it leaves are irrigan Downregulation of llcensing In late G1 phase 1 Prmemsis 2 Yeast e phnsphnlylatinn nl Cdu targets iunr uhig adsln prnleniys39s h Melaznans rphnsphnly aunn nlCd targets iunr prnlenlys39s i z Inhihilnly Phnsphnlylalinn dre onc is phnsphnlylaled hylhe CDKs du ng late G1sGz and sTh39s his is Inading act ily erhnsphnlyimmn dmre onc and Cdu gm DNA during rri39 s39s is degraded d n In ihns cdm quotS Ihelr g late milnsis and early G1 Downregulatlon of licenslng in late G1 phase 3 Nuclearexpnn a 39 7 and cdn h is i cnn act nyleads In nuclearexpnn dinerquot r s nyeasl and melaznans inn nuclearexpn s asrriau rim are i h d n hygerri Ge Ihalhinds cdn hitsl ZEIIV y Lev gsGz an 39nsis lCdI17nn Inadi nlMcm n is degraded during late 39hil n Ilnn n ng milns39s and eady G1 release aquot End of Lecture 4 Molecular Genetics PCB4522 Spring 2007 Lecture 7 chapters 14 continued DNA Replication Version 2 Catalytic domain of T7 DNA Polymerase Pruu 39eadmg ext 35539 Right hand Structure synthetic dnmain has 3 parts Klenow fragment DNA pol I Semidiscontinuous replication leading strand synthesis proceeds continuoule 5 t03 in the direction of the replication fork lagging strand synthesizedin the reverse direction as a series offragments which are later joine 39 discontinuous gynthesis still occurs 5 l to 3 mi 5 lagging strand leading strand Version 2 0X174 phage provides a model for leading and lagging strand synthesis There are two types ofDNA replication in 155011 1 lt1gtXl 74 phage each strand syntheslzed separately unidirectional replication fork a synthesls or the r strand to torrn the douplestranded RFforrn serves as a rnodel tor lagging strand synthesls p synthesls or the m strand to torrn slngleestrands tor packaglng into phage particles servers as a rnodel tor leading strand sysntnesls 2r OrlC origin or bacterial chromosomal replication poth str hesized at the sarne tirne bldlrectlonal replication fork DX174 phage as a simple model for replication Lagging strandquot strand Replicative form RF ds plasmid strand packaged to form vinon l Formation nfthe ths arnndel fur lawgstxan syn ests trcle z Rulllng c replicaann is arnndel d synthesis qgtgtlt174 pnage roiling circle replication provides model tor leading strano synthesis RF V quot d y k 3 ssa o 4 Rep hail2352 Egt Egt W Narmaiivr the r strand is not nicked Removal of the nicked strand Two kinds of activities are needed to convert doublerstranded RF DNA to singierstranded DNA wrtnout synthesis o DNA Helicase separates tne strands uSing ATP to provide tne e srngiestrano binding protein 888 Version 2 X174 Rolling circle replication39 GeneA REF 4 O pvulem Proteins needed for rolling circle replic 39 39 Gene n u 8 m 18 max a a rigi pas Covalentiy linked to o SB protein to keep DNA singierstranded Binding rs highly cooperative Rep protein provides neircase mnctron DNA poi iii hoioenzyme SSB single strand binding protein SSB is a 74 kDa tetrarner protein that binds single strand DNA in a cooperative fashion wrote This protein does not unwind DNA it only stabilizes the single strand form of DNA Version 2 Noncooperative binding O miss DNA WE W gt b0 O H fol low concentration higher 39 NOTE SSE is actually a tetramer Cooperative binding protein 0 Wis DNA km Cooperative binding cooperative binding Sshaped cmve quotn Binding exponential curve cone of protein Cooperative binding pmten 0 W35 DNA M 39 s D W quote a t 3quot J J O higher concentration low concentration Implications most SSB will bind to ssDNA that already has SSB bound tie active replication forks Version 2 Synthesis of doublestranded DX174 RF DNA serves as model for lagging strand syn hesis J Tne pnineiseiine assembles at tne pnineiseiine assembly site pas pax forms hairpin NECWT s DnaE intein o 6 PM is ieeiiinee b Piiinase YEEYUNEd 39 PiiA DnaEisa byDnaEhelicase 0 neiieasetu pvupEl O D We pHmDSDNE me u 553 is displaced PiiAWiineinei hypnApmm pmtEms bmd tu me pas O ase D a PnABCWT syntnesizes sneirt recognition complex RNA WW5 Components of the DX174 Primosome PnA PnAEcamT PnB DnaE piutein pr c is YEEYUNEd by DH 5T PiiA DnaE is a neiiease tn pvupEl DH 53 We piiinusuine Dn ac Roles of Pn39A Trans ueates alung tne ssDNA tei iueate tne pnining site pas by dislucatmg 559 Tnis is amually a neiiease activity Luads DnaE eintei tne urigin Synthesis of doublestranded 0x174 RF DNA serves as model for lagging strand synthesis Shun R NApllmels synthesized in uppusne direction mm pllmusume movement ATP g Primers extended by DNA pol lll Movement olpnmosome AMP RF X174 m PNApllmels removed lowed by Ligase Version 2 X174 priming reaction DnaB protein provides motive force that propels the replication fork l DnaB helicase moves 513 330 kDahexamer DriaB is loaded by the Pri proteins A B C not shown Activates primase DriaG 2 DnaC acts WithDriaB 6 monomers 29 kDa 9 Dnthexamer 5 Du i P39 a 5 a ATP ATP u d is se a 53Egt3AMPgt during loading X174 priming reaction DrlaG RNA primer synthesis 60 kDa DrlaG is a special RNA polymerase used only in DNA replication Activated by DrlaB 5 DnaG associates dinettan or prlmusume movement is 5 El 3 transienth with primosome Version 2 General DNA replication The primosome moves behind the replication fork periodically initiating synthesis of the lagging strand Okazaki fmgments It moves in the anti elongation direction The primase for both CDX174 and E coli oriC priming is DnaG Role of DnaB protein 1 5 to 3 helicase activity generates movement of the rep fork and 2 it activates DnaG Semidiscontinuous r Strand 5 replication tempiaiefor 3 5 lagging 5 leading ieading strand Semidiscontinuous replication The lagging strand fragments are known as Okazaki fragments Usually 1000 to 2000 bases in length 3 lagging strand 1 RNA primer 1112 bases RNA polymerasednaG primase comp lementary to template r DNA synthesis always from 5 to 3 product lagging RF mm and Jr Product leading trand 5 Template strand in RF Version 2 Rmmmna he tennlete smnd t gt5 3 RF Why does mm use a repllcatlon rnecnanlsrn wnere tne repllcatlon fork moves m tne opposlte dlrectlon rrorn tne dlrectlon of Synthesls ottne new strand Orputanomerway Why doesn t Must use a leadlng strand rnecnanlsrn tor botn tne 7 e and e strands J Why lstnls nut an uptlun7 RF Dlrectlun at nellcase ls 5 tn 3 un tne template strand A trequlres pnrnlng by 3 to 5 on tne ar s template Replication of E wu39 Version 2 Replication requires DNA Polymerase Ill 1 w i m i a a catalytic core g subunit b a dimerizauon component that links two cores DNA d a clamp on DNA Furms nanA a 25w proofreading 10 kD 1cm kD catalytic maintains dimen39c structure Forms of DNA p olymemse III rom b xochemxcal smdxes ATP is used Holoenzyme only me n Clzmrl lnzdu mm Vers on 2 Assembly stages fur PanI hnlnenzyme mm GD 0 ATP gt ADP p 155 the clamp loader I d Mumer hlndsln dsDMA quotEMSquot F quotquot m hlnds nun rnl m cm Q Wmm 2 subumzs are encoded by the same DNA7 Holoenxym A tau 1 and a same DNA u dimevmakes The Clamp hu uenzvme mew pvucesswe u dimevbuundtu DNA nuiemeeaiune subunituf DNA ui m head man dimer immeneuneeiapee assemb v m iemuva iemee enemv 15 m mum new DNA pol III events at the replication fork Ful m dnaB helicase DNA pol Ill events at the replication fork O me p meee laws We cammex DNA pol III events at the replication fork new primer synthesized 3 clamp lander Version 2 DNA pol III events at the replication fork 3 Egt V 5 I Clamp uader displaces Pumase Q andthen in 5 clamp mm M r 4 sue ATPuADP DNA pol III events at the replication fork 3 Egt 3 Pm m cumdesvwm me prevmus Okazakw ragment and releases cuntactthh me p clamp DNA pol Ill events at the replication fork gt Version 2 PM Hi dismauesthe da omen Wm em DNA pol Ill events at the replication fork 9 gt ciamp aadev binds anmhev in damp a Prepare mime next mund m inmater The cvcie m Lagging nlE Mime minimum 7312 W 1 u Md IAmvales Pumase Diva e Rennsnnes Byrlass j template Damage 1 aura l Replication notes DnaE hehcasE acuvates PumasE DnaG bv cumac anmg mnew Okazak Th2 Beta u Hemp s uaded nvme gamma 1 cummex mmzme ATP The Beta Hemp mnesm dsDNA Wh2n DNA pm m vuns mm me nex okazak vagment n ve eases n mm a q mn cennecuenswnmau 1 DNA new m we wnesname ma 3 new au mg suanewaemem h n 2M2 eadmu m a mg wand Wmheswsms 3 Emmy abuns and vemma esmnhev upsueam WW2 me we puwmevase cave and hehcase vemam eneaeee RepsI m mvoved m fmsmng aggmg Strand syntheSs DNApul 1pa1A sLarIs synthesis atLhemckbetween DNA and RNA and 55339 the RNApnmer and replaces mm DNA DNA p ul I lzggngstxand Z mck RNApnmEr 1 Sv p p W mek txanslanun 3 DNA ligase l LigaseATP complex formed 2 The ATP changes to AMP as is releases pyrophosphate PP and covalently attaches to the phosphate of the 5 end of the DNA 3 Original phosphate at the 5 end covalently joins the OH ofthe 3 end and AMP is released NOTE T4 phage llgase usesATF E coll llgase uses NAD Version 2 Ligation Enzyme AMP complex Q Q 9 DNAligase fpolNADorRTP Q o T4phagehgaseusesATP OVPVO E 511 ligase uses NAD 320H 0 5 3 Ligase AMP OK I Pt Phosphodjester bond 3 o 5 3 3 5 Ligase seals nicks in DNA Creating replication forks at oriC 1 the strands melt at the origin over a short distance 2 DNA is unwound 3 first nucleotides synthesized as RNA primer Occurs only once for leading strandmany times for lagging strand Exception the leading strand is repnmed at lesluns Version 2 Creating replication forks at oriC 1 Binding of DnaA to four 9 bp sites at on right side of the origin region 2 2040 DnaA monomers form aggregate cooperatively and DNA melts at the three 13 bp sites on the left side 3 DnaBanaC joins the complex to form a bidirectional replication fork Minimal on39C DnaA binding CDC Region ornielting O a L M R 1 2 3 4 ltgt 137mers 9rmers Iq 245bp p GATCTNTTNTTI39T TTATNCANA Note GATC is aDam melhylation site Mutanons DnaAr replication asynchronous mommmers Minimal onC bind cooperatively DnaA protein 4 9rmer sites F AMF DnaBDnao gtDnaB hexamers o a e DNA strands melted at 137mer sites Methylation state of DNA may regulate replication Me Active I origin GATE Active ri in GATC CTAG ale g methylated DNA GAT C Inactive CT G origin T AG Me replica on a Dam methylase methylated DNA e hemimethylated DNA Version 2 Methylation state of DNA may regulate replication nnn hemimethylated DNA Inactive origin Dam methylase delayed 13 min vs lt 5 ml 1 A Me DnaA protein also has delayed methylatjon TC Active origin Creating replication forks at an origin in vitro two other proteins are involved in replication 1 Gyrase acts as a swivel allowing one strand to rotate around the other 2 HU HUllHUZ general DNA double stranded amp single stranded binding protein Bends DNA structural role Similar to histone cooperativity in binding Causes DNA to bend and fold into structure resembling beaded chromatin Molecular Genetics PCB4522 Spring 2007 Dr Eva Czarnecka Verner Lecture 1314 Transposition Genes VIII Chapter 16 Transposons Transposable elemenTs discreTe sequences in The genome ThaT are mobile able To TransporT Themselves To oTher locaTions wiThin The genome quot 1 Move direchy from one siTe in The genome To anoTher do noT need oTher vecTors 2 InTernal counTerparT To vecTors ThaT move sequences beTween genomes phages amp plasmids May provide a major source of muTaTions in The genome 3 Transposons found in boTh prokaryoTes and eukaryoTes 4 TransposiTion does noT rely on any relaTionship beTween sequences aT The donor and recipienT siTes Nonhomologous recombinaTion Ve rsion1 EvoluTion of genomes via 1 AcquisiTion of new sequences horizonTal Transfer 0 geneTic maTerial beTween genomes by exTrachromosomal elemenTs a BacTeria plasmids move by conjugaTion F plasmid Hfr b Phages spread by infecTion c BoTh can Transfer hosT genome wiTh iTs own replicon RearrangemenTs of exisTing sequences Transfer wiThin The genome a Unegual recombinaTion mispairing in homologous recom InaTIon b Nonreciprocal recombinaTion resulTs in duplicaTion of OCI one copy orIgIna unc Ion The oTher evolves c Transposable elemenTs BacTerial Transposons carry genes ThaT Transpose Themselves Similar in eukaryoTes alThough many are defecTive losT abiliTy To Transpose independenle and rely on The enzymes from a few funcTional Transposons TransposiTion evenT may cause deleTions or inversions or lead To movemenT of hosT sequences To new locaTions Transposons may also serve as subsTraTes for cellular recombinaTion sxsTems as porTable regions 0 homologyquot Two copies someTimes on differenT chromosomes in eukaryoTes provide siTes for reciprocal recombinaTion These Types of exchanges lead To deleTions inserTions inversions or TranslocaTions T ansposons selfish DNA concerned only wiTh iTs own propagaTion parasiTe To The genome yeT selecTive advanTage 1 I r are DNA elemenTs ThaT quothopquot or ranspose To differenT Transposon places on The DNA Donor 2 TransposiTion is The movemenT of The Target Transposon 3 This rocess re uires special ProTein facTors l Transposition parTicu arlg To cuT and ligaTe he DNA Transposase 39 4 NO homolo lgy is required beTween The ransposon and The TargeT sequence ll 5 FirsT idenTified in bacTerial operons Transposon silencing 1 BacTerial Transposons conTain inverTed Terminal repeaTs aT eiTher end 2 The Transposase is encoded by sequences wiThin The Transposon i e beTween The inverTed repeaTs 7 Tra nsposase K RecogniTion of The ends criTical n TransposiTion poinT muTaTions aborT IT Version1 1 The smallest Inser ion se enc S em n if RrA huemryire39 809 IR inverted repeat 2000 bp 4obp 2 Most bacteria contain several I elements nd o ten more t one copy on the chromosome Target DNA 3 During Inserj Ion a small Iofn o t e ar e se uence is dlplicatled 4 Sequence of DRs Direct vary between repeat I ength fgnstant for particqu an TCTCAC Acmmc SIP ORFB All 155 contain one OkFTNP 151 is an exce 39 CCTTAA TTGCATAT l39 Transposition v re eat lrbp target eiw P Transposons have inverted terminal re eats and create direct re ea s of flanking NA at the targe si e Most common len th for direct repeats is 9 bp Note 1 target sites are only from 413 bp 2 targets may be random hotspots or preferred bent DNA consensus sequence nactive region H 16 3 5272 v11 1 Rate of transposition 105 1077 per generation element 10 610 lo per generation 10 3104 per element per generation 2 Rate of spontaneous mutation 3 Rate of reversion by precise excision of the IS 103 times less frequent than insertion 2 IS elements of file same type can form a larger transposon called a composite transposon l550 7 Kan39 Ble39 SLIquot 550 7 ilt 7 gti 1 gti Central region carries 1500 l3 drug resistance eemen s rm R Cami IS TS 768 I IS eiemems as direct IS eiemems as direct repeaisArm L repeaisArm R 4757707 m ISIO l 39 i l400 bp Composite transposons ly carry genes for antibiotic resistance or at er markers IS elements only carry enzymes needed for transposI I transposase resolvase IS modules ident cal both functional Tn903 or nosey related differ in functional ability Tn10 Tn5 Note transposons carry ng W 163 resistance markers are 5W Vin desiqnated Tnquot E 1 area direct gtquotW59f39 gt rs quotmm rr ME 4m p m rs mm D Mm r Munniuunb lw 1 A functional IS module can transpose either itself or the entire transposon 2 Composite transposons evolved when two originally independent units associated with the central region IS element transposed to a recipient site close to a donor site 3 Two identical modules may remain identical or diverge 4 Lack of selective pressure of both modules to remain active Version1 A functional IS module can transpose independently OR as a composite transposon unit Transposage i i A IR39 i IR IR lIR 3E1 tnp lllllmmulll tnP ME 7 V g target DNA l 1 y m tnp quotmammalquot tnp m l Two 1510 modules can mobilize any region of DNA that lies Tet ISIOL ISIOR 151m Tel39 1510p Outcome 1 Transposon ntegraies inlu l c rcular DNA Transposon moves again 31221 Outcomez New transposon created by 151w 151w mobihzation onsw modoie n alternative orientation Hg 1M 52 V11 Summary on Transposons 1 The smallest transposons are called insertion sequences IS elements 2 IS elements have inverted repeats at either end and a transposase gene in between they o NOT have any resistance or other markers 3 Two IS elements flanking a marker genes form a composite transposon 4 Noncomposite transposons can either be an IS element or have a single pair of IR39s flanking enzymes for transposition plus other markers Molecular mechanism of transposition I Direct repeats are we generated by introduction 1 W staggered cuts whose lllv protruding ends are wwwm 3539 le moan oi Wyn linked to the transposon The stogger between the cuts determines the length the direct repeats naps at mva w The target repeat is 39 mm rt characteristic of each transposon re ects the geometry o the cutting enzyme Fig lb 5 5212 v11 Molecular mechanism of transposition I Recombination using cellular enzymes I host DNA quot when a Tn inserts a copy at a 151 second site near its orig naI i locat39on between two copies of the transposon singie copy on chromosome Direct repeats result in excision of the transposon or any material between direct repeats Focrxnbrqhvi irmm meniath Woo Fig 16 AZ eerie V11 Molecular mechanism of transposition I Recombination using cellular enzymes mm result in mversmn of the transposon interim mpml Inwma mom on Excissions not supported by Tn39s Precise removes transposo one copy of dupi cate N Vi sequencerare Tn 1o 10 WWW W ma Im precise leaves a remnant of 5272 VII the transposon Tn10 1o39i Version1 The use of Sfuggered ends is common 10 a Molecular mechanism of l l cll39lSPOSl l39lOl39l I transpositions three types M i 1 Replicative M T3lll quot39 35232 a Trans oson is do Icated a copy of the original element is made at a P 39m We 7 recipient site n onor keeps original copy 1 Enzyme 9 b Transposition an increase in the number of Tn copies frunsposase Transeslrificaliori Vi c ENZs transposase acts on the ends of orig nal Tn and resolvase acts Wm M mm M u H W k W on the dupl cated copies 2 Bofh grands dwmy Wad m2 39 39 lar elDNA Within 2 NOYWP39IC llhve transpose nucleoproieinconplexyi a Transposon moves from one site to another and is conserved breaks in donor repa red 3 Transposon leaves donor DNA9 only one b IS and TnlO and Tn5 use this mechanism no Tn copy increase c ENE only transposase 3 Conservative nonreplicative copy o a Tn excised from donor and nserted in target every nucleotide bond is transposon exits conserved like n lambda integration large elements episomes L b ENZs transposase related to Aintegrase family Breakage amp WWWquot iii ini iiivai i iliwliriwi Molecular mechanism of Transposition II Molecular mechanism of Transposition II Replicative 1 transposase Replicative 239 Li9 n m 399 r ends transposition franSPOSi Oquot Strand transfer complexcrossover complex 8 ngallmrl m liil rnds lo ldlgk l eiiils Tl Staggered cuts 5 hp cipan lll target DNA Sillglwill dlltl CUlS ll outside 0nd at Tln Note the cuts are on different strands Demil nt l1l 111 ItLls iiriiiiu ropiimlioii V llimiigli riie Molecular mechanism of transposition II Molecular mechanism of transposition II Replicative 4 Formation of cointegrate Replicative 3 339 ends prime replication transposmon and resolution resolvase TnpR transposition Uses hostcoded functions Fuslun uflwu original molecules requires nosi reolieaiion funclluns Cointegralc Tvvo eooies uflranspusun ms crossover formed by iransoosase resolves to The crossover structure con a ns a ingle stranded region at siaggered ends Pseudrepl cation forks that dupllcale D igi iiiggl39nciii i ii iim li si DNA each of the providetemplateforDNA sy t e inlulargel Homologous Ecnm inacioii al re II integrate Verstom proceeds through a coinfzgr39afz Transpusntun may mse adanar and rectptem rephcun mu c1 cutmegrme pesatmtun reteases twa rephcunsreach as py atthetrarspasan tnmhnagtabhhn recombmatton Molecular mechanism of Trans osifion II Common Infzrmzdlafzs for Transposlhon Replicahvz r quotSP S3939 quot Both rephcattve and nanerephcattve transpastttan use a common mechamsm IS etements praharyattc amp euharyattc transpasans and Replicahvz bactertaphage Mu fransposifion Msor retravtrat DNA and the hrst stages at Common infzrmzdiafzs for fransposif ion Joining transpasah to its target 1 El 1 Synapsts stagzr two ends at 1 L transpasan are brought together y M 2 Transposon ntched at both end Ill target ntched at both strands Ntckzd 2n osswtsz39 c ent cannecttan between the transpasan and the target dsJomzdcr lt amp Common infzrmzdiafzs for fransposifion n1 humusManhattanquot nanspasnanaanng 1m sync m1quot at Wm 1y quot11m news A Mu transpasan passes through a stabte stages 7 MM bmds ta ends as tetramer formtng a synapsts ubuntts act 1n transto cut 7 MM s next to R1 and L1 humatnatetwwa actweme tananpatate DNA 7 Mu acts m transta cut the target stte DNA and medtate 2 g m trans strand transfer 2255 1 d x E Commaquot fnfzrmzd39afzs for 3 Common Infzrmzdlafzs for Transposlhon gin mm d t ansfer39 complex transpasan 1s connected to the target stte through one strand at each end Next stag d39 jgrs and determines the me of mnsEosition Strand transfer comple reahage d reuntan g 151 671 rm fransposifion target nd 1 Mat sthe recambtnattan acttvtty ofMuA m t 1t1 m t M A ctears 5 ma L M an m ltgt Rzphcanvz 41 s n gamma v Nonrrzphcanvz Breakage amp rzumon Vers om Molecular Genetics PCB4522 Spring 2004 Lecture 4Replicati0npart C Dr Eva Czarnecka Verner Course web page httpPCB4522IFASUFLEDU or go to Microbiology 8 Cell Science home page and look under course in aterlal ltDX174 phage Two types of DNA replication in E coli 17 0x174 phage Each strand synthesized separately unidirectional a synthesis of the strand to form the double stranded RF fo serves as a model rorlagging strand synthesis b synthesis of the strand to form single strands for p k ing into phage partic es serves as I rorleading strand synthesis rolling circle 2 OriC ori n of bacterial chromosome Both strands synthesized at the same time bidirectional lt3 gt IJX174 phage Genes VII chapter 12 and13 Genes VIII chapters 13 amp 14 0X174 phage as a simple model foalr replication ewmaten Lagging RoIIing euvalenlly linked strand circle tn the aid un synthesis replication original strand Replicative Leading genome fem RF strand d5 plasmid synthesis 0 strandpaekaged to form virion Means to amplify the a model original replicon Rolling rcle rep n for lead g strand synthe rDN 0X174 phage as a model for DNA replication RF replicative form h 0 h 0 d l ck d 5 05 m omym e 4 Rephellcase SSB o onlysupereorled Jr F 1310 1 E g 39 39 eded just to separate single strands Nnte39 nnnN i a AARF w DNA to singleslranded DNA without synthesis ofnew DNA an en eliease separates the strands using ATP to provide the ergy b Singlerslrand binding protein SSB to stabilize ssDNA 0X174 phage as a simple model for replication cleavage at unit cleavage at rnultirners Rolling Leadlng circle strand replication synthesis G Unit single Multimeric 3 strand single strand H l G 0 Circular single strand G a Multimeric duplex Egt O 7 phage T4 phage Fig 1 13 G VIII Helicase Enzyme that separates strands of DNA using energy from ATP hydrolysis 17 multimeric often hexameric 2 slides along DNA 3 usually has two conformations one binds ds DNA and second binds ss DNA alternations between them drives the motorthat melts DNA duplex 1 2 ATPs per bp unwound 4 specific polarity of function 5 3 or 3 5 depends on preferred end 5 12 helicases in E coli X174 as a simple model for rolling circle replication 2h 9 Aparoiein 4 Proteins needed for rolling circle replication a g gene A protein 0 Nicks at origin pas o providing 3 OH Relaxase protein that cleaves dsDNA amp binds covalently to 5 end Covalently linked to S end of the displaced strand Seal new fraud ltIgtX174 as a simple model for rolling circle replication phage Aprotein G ltgt ugt cgt cgt b Host Rep protein Virion provides helicase function separates strands c M SSB protein keeps DNA singlestranded Binding is highly cooperative D DNA pol III holoenzyrne elongates 3 end ofthe nick SSB single strand binding protein 1 SSB is a 74 kDa tetramer protein that binds single strand DNA in a cooperative fas ion 2 note This protein does not unwind DNA only stabilizes the single strand form of DN ssDNA prerequisite for priming 3 Quickstop phenotype mutanm Noncooperative binding 0 W35 DNA low concentration higher concentration NOTE SSB is actually atetramer Cooperative binding protein 0 W ss DNA low concentration higher concentration SSBs have affinity ior each other and tar DNA synergistic binding Cooperative binding cooperative binding Sshaped curve exponential curve quotnBinding conc ofprotein Eukaryotlc transcription factors use cooperatiyity HSF Almost an ONOFF switch used by regulatory factors ltDX174 synthesis of dsRF DNA as a model for lagging strand synthesis The primosome assembles at the primosome assembly site pas par site forms hairnin Pr ABcdnaT Prlmaae dnaG g recrulted I t b DnaB pnA DnaB recnlited bv WEC h mge N pnA DnaB i C70 hellcaseto propel O PnA Wit tne prlmosome mac 9 We otner protelrls SSE dlsplaced bindstome bvpriA Erltlcal O Prlmase dnae pas furl all ma Sy tHeSlZeS primosome 5mm RNA primer nAB P CdnaT posnecognition complex Primosome PriABCdnaTDnaBDnaG m G W 4 X174 priming reaction DnaB pi oiein 39 rork l DnaB helicase moves 5 3 and unwinds DNA 330 kDa hexamer DnaB is loaded by dnaC with the help of pri proteins Activates primase DnaG primase 2 DnaC acts with DnaB 6 monomers 29 kDa Cooperative binding low concentration higher concentiation Implications most SSB will bind to ssDNA that already has SSB bound active replication forks 0x174 Synthesis of double stranded RF DNA as a model for lagging strand synthesis nort RNA prim re s e Syntheslzed in Opposite direction trom overall prlmosome movement A v Prlmers extended by AW Q DNA pol Movement otpnmosome RF lt1gtX174 Discontinuous thesis of A primers removed by DNA pol tragm erlts ioined by DNA ligase ltDX174 priming reaction DnaG primase RNA primer synthesis 60 kDa dnaG is a special RNA polymerase used only in DNA replication Activated by DnaB DNA DnaG O 3 5 PolHINprini ase 4 3 3 lt2 Synthesis of Dust associates okazak fmgmmt lh Lagging str Lranslen y WI and elongation repllsome ovemmt General DNA replication The primosome moves behind the replisome periodically initiating synthesis of the lagging strand Okazaki fragments It moves in the antielongation direction for lagging strand The primase for both CDX174 and E coli oriC priming is DnaG active component ofthe growing point Role of DnaB protein active component ofthe growing point 1 5 to 3 helicase activity generates movement of the replication fork and 2 it activates DnaG 32 endeffecture 7 Molecular Genetics PCB4522 Spring 2004 Lecture SReplicationpart D Dr Eva Czarnecka Verner Course web page httpPCB4522IFASUFLEDU 0r go to Microbiology 8 Cell Science home page and look under course material Replication of E coli chromosome Replication requires DNA Polymerase Ill 1 DNA polymerase III holoenzyme 900 kDa complex a a catalytic core g subunit b a dimerization component 1 that links two cores c a processivity component E that keeps polymerase on d a clamp loader 1 that places the processivty subunit E on DNA Forms of DNA polymerase 111 from biochemical studies 25 kD proofreading fa 10 kD 130 kD structural holds talltquot together increased processivity Egt 71 kD 1 maintains dimeric structure Forms ofDNA polymerase in from biochemical studies 3930 lt3 Clamp loader 32 kD 52 RD Ammmc a G 7 ya simplex makesp clamp hind tn primedtetnplate lioloerizyme quot6 Emplex mos spar af ATP gt ADP P 9 Assembly stages for P01 11 holoenzyme 1a is the clamp loader is dimer 8 1n recognize primertern plate QM Bw in on 0 ATP gt ADP P aimer clamps on DNA Increased processivity QM Corejoins complex Assembly stages forPol Dlholoenzyme gsAgmmetris nnly nne clamp linden why A Different tforms dimeric alailmesm structure holoemvme dissuciate g Vlhmhz mbum39s neencadedbythesameDNA The clamg Replication of E cali chromosome EgtdnaB helicase Ful lll Replication of E cali chromosome Replication of E 001139 chromosome Replication of E 001139 chromosome Replication of E 001139 chromosome O The ternp1ate for a 1agging DnaG 5 strand is pulled through creating a loop in DNA RNA prim er Replication of E 001139 chromosome The temp1ate for a 1agging 6 strand is pulled through creating a loop in DNA Replication of E 001139 chromosome 2111 Core Pol III The loop is released a 7 1st CorePolIII Replication of E 001139 chromosome New a clamp 8 Core Pol III present on DNA Organization of the oriC Replication Fork e ATP gMP DnaB primase stimulated Note the loading What is responsible for recognizing the sites for initiating synthesis of Okazaki fragments by DnaB Dual properties of dnaB helicase of DnaB helicase by Dnac only 1 Propels the rephcatlon fork occurs at the 2 Interacts with dnaG primase at a correct origin Site 3 5 fragment 39 39 Schematic of Sem1d1scont1nuous repl1cat1on OHC anosome The lagging strand fragments are known as Okazaki fragments Usually 1000 to 2000 bases in length 5 Storey nuuuuuuuuuuuuuuu quot 3 GAppps39 laggingstrand 1 2 RNA primer N1 112 bases RNA polymerasednaG primase one side of the replication fork directed synthesis Related m At ariC the primosome consists of DnaB and DnaG Role of DnaB 1 propels the replication fork through its helicase activity 2 required to activate primase DnaG OriC Primosome directed synthesis 1 binds dnaB to attach pol 111 core to replication fork 1 Speed of Role of Pol 111 DNA 1 Synthesis of leading synthzsilso strand Increase x 2 synthesis of lagging leadiumd strand by extending the mm falling RNA primer Displaces offincreased primase rocessivity 3 pulls the lagging q strand template through the DIE holoenzyme Prlmase Eukaryotic DNA polymerases PRIMASE DNA Pol alprimase complex bifunctional Heterotetrameric phosphoprotein 48 kDa PRIMAS E initiates DNA synthesis makes complementary RNA primer 58 kDa protein tethers primase to 180 kDa subunit 180 kDa polymerase A subunit extends RNA prlmer by making a short DNAi 34 bases only in DraSaphlla has proofreading activity 70 kDa subunit no known catalytic function may recrult poloL pnmase to the repllcatlon fork 000 Eukaryotic DNA Replication 1 DNA pol on I 2 primase subunits initiate synthesis of lagging and leading strands RNA DNAi 34 b primer 2 DNA pol 5 HI elongates both lagging and leading strands 3 PCNA proliferating cell nuclear antigen acts as processivity factor for leading strand like i clamp elongation 4 Replication factors C RFC clamp loading ATPase and RFA single strand binding 5 Topoisomerases I amp H maintains DNA winding Similar functions at bacterial and mammalian replication forks Function E coli HeLaISVAO lieliease DnaB T antigen 1oading helicase Dnac T antigen single strand SSB RFA priming DnaG Pol 7l2prlmases sliding clamp p PCNA clamp loading ya RFC catalysis P01 111 core P016011 holoenzyme I 7 dimen39zation RNA removal PolI MFl exonuclease ligation ligase ligase 1 Origins l ColEl RNA H acm as primer 2 CIDX174 replicative form 3 oriC hairpin CulEl plasmid OXIM RNA ll 4 o pnmosome v Creating replication forks at oriC l the strands melt at the origin over a short distance 2 DNA is unwound 3 first nucleotides synthesized into RNA primer Occurs only once for leading strandmany times for lagging strand Creating replication forks at oriC 1 Binding of DnaA to four 9 bp sites at on right side of the origin 2 2040 DnaA monomers form aggregate cooperatively and DNA melts at the ee 13 bp sites on the left side 3 DnaBDnaC joins the complex to form bidirectional replication forks Minimal oriC O DnaAbinding CDO Region ofmelLing O amp L M R 1 2 3 4 isemers 9rmers I 245 bp p GATCTNTTNTTTT TTATNCANA Note GATC is Dam melhylau39on site Mutations DnaAr Minimal oriC 20740 monomers bind cooperatively DnaA protein Does DnaA act as origins Vs cell mass H DNA strands 4 9mm sites melted at 137mmquot ATP AMP 39 DnaBDnaCgtDnaB hexamers a a Methylation state of DNA may regulate replication Me Active I origin GATC Active CTAG origin I e replica on a Dam methylase GATC methylatedDNA CTAG M g methylatedDNA GATC inactive CTAG origin Me hemimethylated DNA Methylation state of DNA may regulate replication Membranerb ound inhibitorr competes with DnaA for onC V e GATCnn nnnnnnnn h th I t d DNA CTAGnnnnnnnnnn am y Inactive origin Dam methylase delayed 13 min vs lt1 5 min owl promoter repressed Me DnaA protein also has delayed methylation Active origin Creating replication forks at an origin in vitro two other proteins are involved in replication 1 Gyrase acts as a swivel allowing one strand to rotate around the other 2 HU HUIHUZ general DNA double stranded amp single stranded binding protein Bends DNA structural role Similar to histones m cooperativity in binding Causes DNA to bend and fold into structure resembling beaded chromatin Molecular Genetics PCB4522 Spring 2007 Lecture 3 Conjugation Genes VIII Chapter 13 Course web page httppcb4522ifasufledu or go to Microbiology 8 Cell Science home page and look under course material The F plasmid is transferred by conjugation between bacteria 1 Bacterial conjugation a plasmid genome or m chromosome is trans erred from one bacterium to another in a mating process mediated by F plasmid 2 Fplasmid an example ofan episome in E coli 3 Episome an element that may exist as a free circular plasmid or that may become integrated into the bacterial chromosome as a liner sequence Bacterial conjugation means to exchange genetic material between bacteria Connects replication With the propagation of the netic unit Fplasmid 1 large circular plasmid 100 kb 2 only 60 ca 60 genes has been mapped 3 32 kb is organized as a unit to transfer is genome to another bacteria transfer region or Ira genes 4 two methods of replication a oriV as free plasmid one copy bacterial chromosome b uses E coli chromosomal origin when integrated oriC oriV is suppressed FPlasmld Discrete region that has transrer genes oriT Origin oftmnsfeo tra amp trb loci 40 genes used to initiate replication for transfer m genes IS elements insertion sequencesusedin transposition Fpiasmid integrated into cnromosomein rare instances two mechanisms oriV usedto Inmate 1h mo g us plasrniol replication recombination 2 transposition F 1 d Hfrcell contains 3913 351111 integrated F plasmid F cell contains episomai F plasmid E ml chromosome After integration Fplasmid replicates as part ofhost replicon 0 1C miV is suppressed Chromosome Transfer formation of Hfr strains high frequency recombination The Sex Pilus Some E coli 5 strains contain There are Mo f qtilityplasmids r mechanisms of 3 A integration I Carries the orquot 09 information mom ma 390quot F CELL lptqggriquuai required for its 2 Transposition l mtransfer Depending onthe 39 MFplasm39rt DNA Is NOT integrationthereare 39 q d ui erent Htr strains quotll CELL e pilus Single stranded genomes are generated for bacterial conjugation tra region of the F plasmid regulation 1 Fplasmid is 100kb amp takes about 5 min to transfer 2 Sequences required for transfer are located within a 333kb transfer region IncF1 3 Pilus synthesis and assembly Nicking amp initiation of transfer mating pair pore formation transfer of DNA matingaggregate stabilization surface exclusion regulation O traJ auwaur traVl nirecoon Transcription unit a trendy Egt Q oriT IlaMJ YALEKBPVRC WU N 1111an vaFrrbB lidl G STDIZ l in 7 Ira amp Irb loci 40 genes Expressed coordinater as a part of singletranscription unittraVtral Hg 13 20 Gene VIII Fplasmid 5 Fpositive F bacteria are able to con39ugate mate with Fminus F39 bacteria 6 In its integrated form the Fplasmid may cause some or all of the bacterial chromosome to transfer to the Fminus recipient 7 Fpositive bacteria possess pili formed from the pilin protein FPilus 1 Extracellular filament that extends from surface 23 pm hairlike 23 pilicell main structural component is a single subunit pilin coded by MA gene tip protein 2 Pilus recognizes various receptors on the host cell mating pair formation may occur differently on solid or liqui me ia r n are Essential nt in 21mg nainnn but are NOT channels int DNA transnnn Conjugation 1 tip ofthe Fpi1iis makes contact with recipient cell a piliis is composed ofpilin siihiinits which form a hollow cylinder ofs nm wi 39 diameter not DNA does NOT transfer Lhroughthl tube b 1fpotentia1 recipient is Fpositiye no connection is formed due to siirhce exclusion proteins coded by traS and traTofFplasmid Conj ugation z piiiis retracts bnngmg recipient eieser for transfer 3 DNA transferred Lhmugh channel fanned by protein ended by trap gene TraN and TraG may alsu participate in pure fairnatian 4 Transfer hegins 39um anT which is nicked hy TraYTral cumplex at a me site Trai actuany nicks 5 TraYTral rnidtirnene cumplex migrates around circle and unwinds DNA Gum 539 end at ca lZUprsec a Only one unitiengthistransferred Mg 13 22 Gene VIII Overview of Conjugafion 1 Comugaiivz Piasmids are either szifriransmissibiz or biz mobihza 2 The transfer systems are encoded by the trc genes contained on the piasmids themseiyes a Tra systems are iinhed to their incompatibiiity Inc group Friypz Incf RN piasmids 91nd 4 Piasmids that have tranfer systems that aHow transfer of D A to unreiated species are hnown as gromiscuous piasmids Incw piasmids Incppiasmids aIncN Mechanism of self Dunw m5 iamviml transmissible transfer Q 1 A site nnthe piasmia knuwn as the EINgm nftransfer 1777 is Manna weir n cked by aspecific endanuciease TraI Trav is aisn a part nfthe W 0 campiex T strand quotW 3W aispiacemenx 2 A w is farmea between the twa ceiis aria anty or strand af 1 3 DNA is passeathrnugh tn the ether ceii 5 and first may er C he s n ie strana h each ceii oniy Ingleunll undergues rephcmiun to term gmggmw semmn aaubie stranded DNA O C F Mechanism of plasmid obilization l The mob piasmid cannot transfer without another piasmid inn nnienanr 2 The other piasmid heiper gamma piasmid ma or ma not be a sei iransmissib z piasmid U but MUST CONGWV Va quot W Eh39 functions ceii contact 2125 niching tins 3 1f the heiper piasmid is aisoiiaiai sei iransmissib z it may in aiso transfer i Major Functions During Transfer gt40 genes rav binds near oriT and recruits traI reiaxase like A protein of c1gtgtlt174 2 TroI has nuclease a hehcase ATP activity Function enhanced by Trav a IHF integration host factor Tr S a transferase 9 covalent attachment of the 539 end of the DNA to the protein Trab active transport binds DNA ATPGTP binding sites inner membrane protein necessary for DNA transfer Surface Exclusion Reduces conjugation among cells carrying closely related plasmids 5 exclusion groups identified date 1 TraT outer membrane protein that blocks matingpair formation 2 TraS bocks DNA transfer Chromosome Transfer Hfr 9 Recipient cell Part1 at c r rneurnoi roii rim omoi rmoinosone is V 7 rneirirer re sotcir Brnkago donoi DNA strand Chromosome Transfer Hfr 9 Recipient cell Part 2 rne oonai one icsinieni ociisrcoaiaic usoaiir Detail oniiio an no t 9 in aroonnt cii and donor ceii are mad oouotr wonder l campiensnnir to ins onimnsioiioa some at one and renrins nii seii DNA integration oi donor chromosomal DNA Chromosome transfer sfer process uses the rolling circle method of replicatrori The complement to the transferred strand is synthesized in the recipient It takes I min to transfer entrre chromosome ufE call The douiole stranded transferred DNAls integratedirito the recipient chromosome ioy douiole recomioirratrorr 3 repositiye strains supporthigh levels ofrecomioirratiorr and e descri ed as HR strains high 39equmcy of recomioiriatrori Chromosome transfer 4 The transfer ofthe host chromosome is away from the tra region and Fplasmid except for a small art around 0117 5 Typically only relatively short stretches ofDNA are transferred amp are integrated into the recipient Chromosome transfer 5 chrornosorne transfer usually does Lotresult in conversion ofrecipient ce11 to Ft 7 1n chrornosorne transfer donor DNA integrates into the host genorne by recorn 39na 39on or os on 1n plasmid transfer this does not occur Chromosome transfer 8 Bacterial contact usually broken before DNA transfer complete A gradient oftransfer frequencies around the chrornosorne 9 E cch chrornosorne as amap divided into 100 minutes the starting point for the gradient oftransfer is different for each Hfr strain detennined by the F factor integration site B m Formation of Prime Factor Plasmids Hfr Cell F39 Cell Inlegraled F F39 plasmid plasmld Chmmosome Chromosome 1 Plasmlds that leave the genome carrlelg chromosomal NA are known as grlme factors 2 They leave the chromosome by homologous recomblrlatlorl resultlrlg m a deletlorl m the chromosome tra region of the F plasmid regqu n M newteetultshaltus ehdutDNA Iralrcuvalenlly attachestu 5 end utDNA amp uhwmds lt IraYIral Champ nun nickmg nd unwrnding onTtraMJ YALEKBPVRC WUN trhCDE traF trbB traH G 5n Ia mP sat a uhaetlch m hamster rilin IraTruutetmembtarle 5m plutelnthatblucks mumquot Ir Fur 1mm lino Irasblucks DNA lml Heine IirP tlanstel can m Bacterial replication is connected to the cell cycle chapta39 13 14 Genes Vlll 1 two links between reglication andbacterial cell growth a Frequency of initiation adjusted to t rate ce11 is growing h The completion ofa rephcation cyc1e is connected to cell division Connecting bacteIial replication to the cell cycle 2 Doubhng time time needed for ce11 numbers to double In E can they range from 18 rnin to slower than 180 min average in lab is 60 rnin Connecting bacterial replication to the cell cycle 3 Constants in replication cycle 2 c xed time 40 min to replicate entire chromosome Replication fork moves at ca 50000 bpmin g D xed time 20 min between end of replication and cell division 2 c 60 min These values apply for doubling times between 18 and 60 min Connecting bacterial replication to the cell cycle 3 Constants in replication cycle continued d When doubling time is less than 60 min i ati n ofreplication must occur before n the end ofpreVlous lelslon cycle Connecting bacterial replication to the cell cycle Example cisi iiiiinias sierst nannies Fig124 rneman nits IM su mm es n inniniiin in reiiiicniiin nii sis n m 5 can is nie iii iiiiinie eiisiy in minutes nun now isinis usable llllllnn occurs on or ns iin ins chromosome unvnenpnnrn on rmwivln minersquot areshmlm here How does the cell know When to initiate e replication cycle Rapidly growing cells are larger and possess a greater number of origins multiforked chromosomes Initiation occurs at aconstant ratio ofcell mass to the number ofchromosome origins a Bacterium unit cell an entity 17 m long one origin perunit cell A rapidly growing cell with two origins will be 1734 nmlong b At 10 min alter division the cell mass increases suf ciently to support an initiation at both origins How is cell mass titrated 1 Current model suggests at39 39tiation is controlled by the accumulation of a positive acting factor not an inhibitor Accumulation of a critical amount would trigger imtiation This factoris dilute in newly divided cells 2 Alternative model suggests that imtiationis controlled by th ac e cumulation ofanegative ac 39 R factor aninhibito 39 39 39 tin r Inhibitor may be synthesized effective level in larger cells initiation Assigned text passages You are responsible fo Chapter 1315 Genes VIII Figs 1325 amp 1326 The septum divides a bacterium into progeny each con a39 ome t Ining a chromos amp Chapter 1321 Genes VIII Figs 1336 1337 amp1338 Single copy plasmids have a partitioning system The end of lecture 3 Molecular Genetics PCB4522 Spring 2007 Lecture 7 chapters 14 continued DNA Replication Right hand StructurF syntthc dnmain has parts Klenow fragment DNA pol I Semidiscontinuous replication readingsixand synthesispmceedscununuuusly s39m 339 m the meeaen quherephcanun nrk iamg strand synthesized in the reverse direchun as a senes Dreamean which arelatenmned This discnn nllmls smthesis SD11 accurs 539 U 39 mi 5 339 539 W 3 and laggng strand leading six v x174 phage provides a m d leading o ei for and lagging strand synthesis etwu types ufDNArephcauunmE call 17 x176 phage each slvand Smihesized sEpaialelV unidirecliunai iEpiicaliun mm a Wnlhesis mm 7 sliand in luvmlhe duubierslvanded RF1mmsewesasamueeivuvmmumwmhesis b Wnlhesisul e a sliandlu 1mm smelesuanesmv PackaginginluphauEpamcles evsasamudei luvleadmgslian wsmhesis ZrOnCuHElnmbacleiiaichmmusumailepiicaliun bumsliandssnlhesizedallhesamelime bidirecliunai vEpiicaliun rum sexm phage as a simple mudel fur replicatiun aaum LE gtO c35 CD strand Replicanve sum dsplasmid strand packaged in farm vmun Catalytic domain of T7 DNA Polymerase Version 2 Xi74 pnage roiiing circie repiication provides rnooei ror ieaoing strand Synthesis RF W W mm ymm ssa o 4 Repweiicase Egt gt g W Narmaiivi tne r Sharia is not nicked Removal of the nicked strand Two kinds of actNities are needed to convert douoiestranoeo RF DNA to singieestranded DNA Witnout Synthesis of new DNA Heiicase separates tne strands using ATP to provide tne singiestrano binding protein 888 X174 Rolling circle replication SSE W o 373 lt it C gt O Proteins needed for rolling circle replica ion ene A protein to nick at origin pas covalently linked to 5 end ofme displaced Strand SB protein to keep DNA singiestranoeo Binding is nigniv Ooperalive c Rep protein provides neiicase mnction DNA poi iii noioenzyme SSB single strand binding protein SSB is a 74 kDa tetrarner protein that binds single strand DNA in a cooperative fashion wrote This protein does not unwind DNA it only stabilizes the single strand form of DNA Noncooperative binding 0 miss DNA WE low concentration higher concentration NOTE SSE is actually a tetramer Cooperative binding protein 0 Es DNA km low concentration higher concentration Cooperative binding coopemtive binding Sshaped curve quotn Binding exponential curve cone of protein Version 2 Cooperative binding protein 0 W35 DNA km low eoneentrabon higher concentration Implications most SSB will bind to ssDNA that already has SSB bound tie active replication forks Synthesis of doublestranded DX174 RF DNA serves as model for lagging strand syn hesis The bnrneiseirne assembles at the prlrnusurne assembly site pas PnAECme naE pintein is ieeidted b Piiinase ieeidited PiiA DnaElsa byDnaaheliease helieasetn pinpel DnaGO D the lemDSDmE too PYlAWlth nthei pmtElns bind to the pas o SSE i displaeed by PiiA pintein 7 F39nrnase D a PnABCdnaT 7 synthesizes shun recognition complex RNA prrnE Components of the 0x174 Primosome Synthesis of double stranded ox174 RF DNA serves as model for lagging strand synthesis PnA p B PnAECme H DnaE pibtein DnaBC pr c is ieeiuited by shun RNA lemEYS D T PiiA DnaElsa synthesizedin uppusne quot5 heliease tn piupel diieetiein tiuin lemDSDmE DH 55 the lemDSDmE D inuyeinent ATP Piiineis extended by Dnac g DNA pnllll Movement ntprimnsnme AM Roles of Pn39A Translucates along the ssDNA tei lbeate the priming site RF ltIgtx174 pas by dislbeating sea This is amually a heliease Discontimwus a lvl t t n e synthesiwthe W ENSiflm39i i39T ea s na on b e Drlgln y no an ianmen s 39 Strand ibined by Ligase X174 Prlmlng reaCllon X174 pnmmg react10n rotein provides motive force that propels the k DhaB p replication or 1 DnaB helicase moves 513 330 kDahexamer DnaB is loaded by the Pri proteins A B C not shown 39 DnaG Activates primase 2 DnaC acts With DnaB 6 monomers 29 kDa DnaB hexamer Dn P DnaG RNA primer synthesis 60 kDa DnaG is a special RNA polymerase used only in DNA replication Activated by DnaB DnaG associates transienth with primosome Orientatan ntternplate direction bit prlrnusurne rnnyernent is 5 to 3 Version 2 General DNA repllcatlon Semidiscontinuous mam The primosome moves behind the replication fork replication template for periodically initiating synthesis of the lagging strand Okazaki fmgments It moves in the anti elongation direction The primase for both CDX174 and E coli oriC priming is DnaG 4 4 3 5 lagging Role of DnaB protein leading l 5 to 3 helicase activity generates movement of the rep fork and 2 it activates DnaG emplatefor leading strand P du t 1 di Semldlscontlnuous repllcatlon X174 an quot5 The lagging strand fragments are known as Okazaki fragments Usually 1000 to 2000 bases in length Template smmd in RF 5 Template strand GAP Ok kl f t 5 1quot gm 5 Product lagging lagging strand 1 2 comilemem Y to and RF x174 RNA primer Nu12 bases mp 5 3 gt RNA polymerasednaG primase DNA synthesis always from 5 to 3 Q Why does mm use a replication mechanism Where the X174 replication fork moyes in the opposite direction from the Rolling circle direction or synthesis or the new strand 0r utanotherway replication WW 09 Srl t itiust use a leading strand mechanism ror both the r and m strands 39 l A treouires priming by W M Smm Primase Primase V contact With DnaB helicase for an option7 E S 9 nellcase Would have to move Rmsllarlmthe ienpisie smnd ards 3 to 5 template orl the Direction of helicase is 5 to 3 on the template strand Version 2 Replication of E coli chromosome Replication requires DNA Polymerase ill 1 DNA polymerase III holoenzyme 900 kDa comp lex a a catalytic core g subunit b a dimerization componmLELhaL links two cores c a processmty component gmat keeps polymerase on DNA d a clamp loader that places the processmty subunit on DNA Furms at DNA pulymemse HI rum biuchemiczil smdies 6 am a proofreading ea 10 kD S NCNI ZI 5 an n catalytic 99m a 71KB increased processiwty maimains dimeric structure Forms of DNA p olymemse III rom biochemical studies 15 cnmpllemds a damp iintii primed tanplate ATP is used Holoenzyme nnly one p Clzmrl loader mm Assembly stages fur PanI huluenzyme Clamp in del is yais the clamp loader mm v m Egt 39 3 WW ATP gt ADP F W Hlmve I d Mimer hindsin dsnun quotEMSquot quot39quotEESSNW mu hinds min ruil Ill cine Q Which 2 subunits are encoded by the same DNA7 Version 2 u dimeimakes DNA pol III The Clamp kuiuenzvme mgkw pvucesswe events at the replication fork u dimeibuundtu DNA butshdesaiung Pal I subunit uf DNA ui m dnaB helicase head m 3H dimer u dimevis NYE shaped assemb v m vemuvai veuuwes enemv 16 rm 141a LW 1 We vm DNA pol III DNA pol III events at the replication fork events at the replication fork 5V newpnmer O me We 1 quot m Jig by we W 2 3 p DNA pol Ill DNA pol Ill events at le replication fork events at the replication fork 339 Egt 339 Egt 5 B 5 ciemp aadev PM Hi mHides Mmhe mma W the 5 mp Version 2 DNA pol Ill events at the replication fork Pai iii aisniaaestne aarin iaaaer rmri tne Okamki tmgri erit DNA pol Ill events at the replication fork 339 Igt ciarin inadevbirids anatner n ciari pt Pvevae tar tne next 6 ram at intietian Tne cvcie cit Lagging Btrang antnesis imiriirrmr IIIH rescue reniieetian rete W t u iaia 39Adivales nririase DriBG J Beniiscrmes Bypass iemniae Damage Replication notes DnaB neiicase activates Brimase DnaG bv cumact Briming U1newOkazakiViagmentsismmated even14 kb Tne Beta n ciamn isicrageg nvtne gamma r cummex utiiizing ATP TneBeta ciamn bindstu DN vvnen DNA rmi iii runs into tne nevtokazakiiragment it reieases trumtne Beta ciampi but is stiii retaineg w tne nuiuenzvmetnrcrugn connectionsWitn tau r DNA rmi iii cure wingsbacktu start a newiagging strangiragment bv gisniacing tne gamma imaging cumniev trumtne Beta ciamn vvneneitneri dm uvaEEmEwandSmmsismsaEsiu in abuns ang reinitiatesiurtner upstream wniie tne citner nuivmerase cure ang neiicase remain engageg RepsI is lnvoved in finisning lagging strand svninesis DNApBl i palA starts synthesis acute m k betwem DNA and RNA and s e exanuclease activity remaires the RNA pnmer and replaces it With DNA 0 DNA p l I laggngstxand Z mck RNApnmer i Sr p p W 3 A mcktxanslauun 3r Version 2 DNA ligase Ligation Emma AMP l LigaseATP complex formed 0 complex 2 The ATP changes to AMP as is releases DNA ligase ingot rATP pyrophosphate PP and covalently O OED 0 gi l fgs gl v attaches to the phosphate of the 5 end of short 5 5 I the DNA 5 3 3 Original phosphate at the 5 end covalently 3 Ligase AMP joins the OH ofthe 3 end and AMP is 0 o P Phosphodiester bond released 5 Q 5 i M T4 phage ligase usEsATF39 E Coliligase uses NAD Ligase seals nicks in DNA Creating replication forks at oriC Creating replication forks at oriC 1 the strands melt at the origin 1 Binding of DnaA to four 9 bp sites over a short distance 2 DNA is unwound at on right side of the origin region 2 2040 DnaA monomers form aggregate cooperatively and DNA melts at the three 13 bp sites on the left side 3 first nucleotides synthesized as RNA primer Occurs only once for leading strandmany times for lagging strand 3 DnaBIDnaC joins the complex to Exception theleading strand isrepnmed atlesiuris form a bidireCtlonal replication fork Minimal on39C Mutations DnaAr replication asynchronous 20740 monomers Mmlmal 0 10 bind cooperatively DnaA binding QC DnaA protein Region or melting L M R 1 2 3 4 DNA strands 137mers 4 9rmer sites melted at 137m P AMP sites a DnaBDnaO gt DnaB he C 9rmers I 245bp GATC TN TTN TTI39T xamers e on TTATNCANA Note GATC is aDam melhylation site Version 2 Methylation state of DNA may regulate replication Me Active 39 origin GATC Active TAG n39 in lxlle replica on a Dam methylase GATC CTAG r39re g methylated DNA GAT C Inactive CT G origin methylated DNA e hemimethylated DNA Methylation state of DNA may regulate replication Membranerboundtnhtbttorr competes wlth DnaA for onC nnnnnnnn nnnnnnnnn hemlmethylated DNA Inactive origin Dam methylase delayed 13 min vs min deA promoter repressed also has delayed methylatjon lt1 5 Me DnaA protein TC CTAG Active origin Creating replication forks at an origin in vitro two other proteins are involved in replication 1 Gyrase acts as a swivel allowing one strand to rotate around the other 2 HU HUrHUZ general DNA double stranded amp single stranded binding protein Bends DNA structural role Similar to histone cooperativity in binding Causes DNA to bend and fold into structure resembling beaded chromatin Transcriptional regulation in EukaryotesMediator Part 3 Alarge complex of proteins wn as the Mediatoris also a component of the Preinitjmjon Complex Mediator The Mediator contains uptostuhunits Note iArsare nntsnnwn g ad Stmcmres M Mediator and A Polymerase ll llalnalzymeiiEEE Francisco i Asiurrase n Wei liangi Lawrence c MyErsi Claas M Gusassnnii Roger D Knrnbeigi science 233 985 gm ne 3 Hulnenwiielnrnzdhvvl Medium and RNAvnlWerase llin pmieernn lnlqesni 73 pamcles were signed and quotmeme calmlale inenrap ueereimrsrn anenended mnlnrnulinn untinead ilmddlemland muse nee we mimannernseeenierenneursneeie Mediator com plex Mediator proteins Bipg rsto CT 2 5m io roiei nereaseinbas transcription in vier phosphorylation of the CTD integrator of positive and negative signals Mediator 4 components SRB proteins suppressor of RNA pol E ii mutations in these genes overcorne mutations in the CTD Tnisirnpliestnat and their functions are unknown cm dunasetal i999 ueeremunnnpni ll A nren resolutionvrmein Vast Mmlamr emu moleic ads Research 2m anion map W e la or subunits can be m biochemically willM l Whig puri ed as modules 39Srb4 39Med9l10 39Gal11 Evimmeinra Medalan mu vuwerase ii rramcnminmi Reguaiinn c sewedimmeslt Henri Marc Enumm Eell ml lt 143451 yeast yeast Head Srb4 module Attacnment sitesto RNApol ii and tire CTD olRNApol ii A mutation in SrbA eliminates 93 ol transcription actuated amp basal middle middle Q head i Middle M edEl module Speciiic function unclear yeast yeast Tail Gal ll module Contacts Witn transactivators and repressor proteins Sin4 mutants f tailless j are delective in ac nated transcription CdklllecC Cdk8Cch SrleSrbll 3rbio CdeSrbii cm module innibitsTFllH by pnospnorylating tne cycH subunit olH Tne cde module is dissociable yeast Mediator has many forms in animals lt nas been proposed tnat tne smaller Mediator complexes ie P02 and CRSP are tne ones tnat activate transcription and tire larger complexes TRARARC StDRlP contain the CdeC cC module and otner subunits tnat innibit transcription 0ne view is tnat tne multiple lorms ol Mediator reiieci responses to coniaci witn dillerent activators tnat are present in various cell types and during development Acrosstalx may ist between tne signals received dtne subunit com osition o Mediator Acompeting possibility is tnattne dillerent lorms ol Mediator are yust artilacts olisolation approacnes ex an namcnuimai reguaiinnmmm Memanr like maaivamm st and melazmn cells thzil Malik nd Ruben a meaerannnnos 25 21er Mediator can be puri ed using an al nity column with 39 b39 d CTD hvsis Yransaawalursdetevmme ADO mm mm m Mmmwm h ratesmmmamnandrelmtlaimn Specmc Memalnv cnnlnvmallnn delevmmerl bvlvanadwalm contact medmurcmdw vcmvlex mum We mles lntnnscn lmzl reguztmn KlananA W5 and mnnvwembew luumal 5 m 2m lznn mu Suence m 3mm mam me that nm alllvansaclwalms cnmacllne Gal 11 tall module Gal 6 mmaassm m me head all Suence m 3557 ms mam smm cm phushuvylateslhe cyclm H subuml u TFllH thus mmmmg TFllH and tvanscvlptlun smm alsu phushuvylateslhe CTD ulpul u beluve quotmam pvevent Jummgt n whlch s pul ll mm he PlC anclivmnr Complexvs mm MWI W commquot www mm can m cu l 3rtrmw 3 cm in 5N3 r V mum 7 cmuumcwm r mum r r w r N 39 Polll m 7 quot7 new 2 39w M t mquw A szslr rlmluns 1 msvupts nuclensnme s1mcluve vesmmg m 3n4nm mcvease m mnmng amansmwalm Pmlems 2 lemme ATP a mu cna lvalnls amelvlates nls1nnes Holoenzme mammalian cells Contains mediator plus most basal transcription factors except TFllD and TFIIE Mediator and holoenzyme Molecular Genetics PCB4522 Spring 2004 Lecture 2 Replication Dr Eva Czarnecka Verner Course web page httpPCB4522IFASUFLEDU Dr 90 to Microbiology 8 Cell Science home page and look under course material Identi cation of protein components involved in DNA synthesis 1 temperature sensitive mutants conditional lethal mutants replication at permissive conditions but fail to function 39 39 conditions high temp 42 C In E 601139 identi ed loci dna genes at nonpermlsslve 2 dna genes a quickstop mutanw immediate stop in replication elongation enzymes defective amp defects in precursors b slowstop mutants defective in reinitiation smaller class Chapter 13 DNA Replication Chapter 15 in Gene VI amp14 in VIII Primosome a protein complex that initiates synthesis of a DNA strand Replisome complex of proteins aged in elongation of the newly synthesized DNA strand Assembles at the replication fork Identi cation of protein components involved in DNA synthesis 3 in vitro complementation systems combine extracts from mutant and wildtype strains Can add back purified proteins to identify function of a specific dna gene product Progress much slower in eukaryotes DNA polymerases enzymes that make DNA 1 Both bacteria and eukaryotes contain multiple DNA polymerases 2 The ones that actually replicate the DNA are called DNA replicases 3 All have the same type of synthetic activity a each can extend a DNA chain by adding nucleotides one at atime to a 3 OH end b the choice odeTPs dictated by base pairing with the template strand DNA polymerases enzymes that make DNA 4 Some function as independent enzymes 5 Bacterial DNA replicases contain a large umber of subunits large protein assemblies It is hard to say which proteins are actually subunits and which proteins are just loosely associated Five DNA polymerases in E coli 0 1 poll encoded by polA gene a Major m enzyme for damaged DNA b Plays secondary role in semiconservative replication c Most abundant 400cell d Molecular mass of 103 kD C9 2 pol H encoded by polB gene a Minor DNA repair enzyme b Molecular mass of90 kDa Five DNA polymerases in E coli 3 pol III encoded by polCdnaE gene a REPLICASE39 de novo synthesis of new strands 0 DNA b Contains many subunits c There are 1020cell c Molecular mass of900 kDa c Has no 5 to 3 exonuclease activity Five DNA polymerases in E coli 4 pol IV encoded by dinB gene a SOS repair enzyme ofdamaged DNA 5 pol V encoded by umuD ZC gene a SOS repair enzyme ofdamaged DNA Phage coded DNA polymerases 1 T4 T5 T7 amp SPOl a 5 3 synthetic activities b 3 5 exonuclease proofreading activities 2 Mutations prevent phage development 3 Each phage polymerase peptide associates With other proteins phage or host to make the intact enzyme Eukaryotic DNA polymerases Five identi ed in mammals Enzyme all all 5i B I Location Nuclear Nuclear Nuclear Nuclear Mitochondrial Jnction priming elongation repair amp repair replication ofboth ofboth replicati strands strands 355 No Yes Yes No Yes exonu 0 relative 80 1015 215 activity PRIMASE REPLICASE Eukaryotic DNA polymerases PRIMASE DNA Pol aI39 rimase complex bifunctional Heterotetrameric phosphoprotein o 48 kDa PRIMAS E initiates DNA synthesis makes complementary RNA primer 0 58 kDa protein tethers primase to 180 kDa subunit 180 kDa polymerase A subunit extends RNA Q 39 by making a short DNA only ln Drasaphlla has proofreading actiVity Q 70 kDa subunit no known catalytic function may recruit polo primase to the replication fork Eukaryotic DNA polymerases REPLICASE DNA polymerases 5 III and 5 II 1 Heterodimeric 2 Need auxillary proteins a RFC replication factor C binds to RNADNA primer amp stimulates assembly ofpol 8 or pol s b RFC loads PCNA processivi factor onto DNA PCNA binds to 5 or s amp makes them stable on DNA 3 Intrinsic proofreading activities 4 Probably synthesize all cellular DNA 5 to 3 DNA synthesis occurs by adding dNTPs to the 3 end of the strand 5 PPP 3 OH 0 PP Choice odeTPs GC ArT 6 DNA synthesis has an extraordinary high delity between 398 and Q40 1 error per genome 4200 kb per 1000 bacterial replications Substitutions frame 5 39 7 Proofreading function all bacterial DNA polymerases have a 3 5 exonuclease activity Operates in the reverse direction from synthesis Processlvlty 8 In proofreading the excised base is replaced by a different active site of the enzyme than the one used for the original synthesis Expected error is1 per Proofreading drastically reduces the errors made in replication Enzyme Synthetic Proofreading Errorrate dom 39 omain proof proof DNA polI aa200600 Ntenninal 10395 5 x107 DNA po1 111 o subunit 2 subunit 7 x106 5 x109 T4 DNA po1 ctenninal Ntenninal 5 x105 10397 Rev transcrip none 10395 E 001139 DNA poll polA l easily cleaved into two fragments by proteinase a large fragment Klenow fragment contains olymerase and 3 5 exonuclease proofreading domain Used in vitro for synthesis reactions DNA sequencing Klenow fragment 68 kD srnall fragment 35 kD l C Em C xonuclease polymerase 5 3 exonuclease 3 5 39 site 5 proofreading catalytic in vitro DNA synthesis using the Klenow fragment DNA p01 1 Emerimental User a Fillin reaction to label recessed ends of DNA outlnem b DNA sequencing HP 5 i 4 3 5 Unique ability to start replication at a nick in DNA Klenow fragment DNA pol I Catalytic domain of T7 DNA Polymerase Large 2123 Right hand structure synthetic dnmain has 3 parts DNA pol I pal1 DNA P01 1 pom small fragment has 5 to 3 exonuclease only 2 Nick translation initiates at nicks in D A Extends the 3 H end while removing the strand in front by its 5gt3gt exonuclease aetivity Displaees existing This activity allows pol I intact to be used for nick translation m Vltm 68 kDa 35 kDa Klenuw agnent small fragment N m c m exmdm pulymmse emudease 3 DNA ol I In DNase is used form vttm zws39 suzv labeling ofDNA by nick translation qu 39eadmg dernain Note rerneves RNA pnrner amp 1E hp DNA Nick translation by intact E l DNA 1 I ca 1 0 DNA p01 I p 1 add DNasetnniek DNA es m VZVU function 339 70H 539 VP 5 T Filling in short stretches ofslnglestranded 3v 539 DNA that arise from39 exeised DNA agnents 22 Q 2 a DNA replication lagging strand 3v 339 Add une FrdNTF three enld dNTFs and D vAonl l b DNA m air when damaged bases have note nick moves 5 to 3 been removed E coli DNA pol III Replicase 1 In order to study must use polA mutant strain of E 601139 since the pol I concentration is so great that it verwhelms pol III activity In vitro studies use extracts from polA mutant cells 2 Subunit structure Q a 0L subunit 130 kDa DNA synthetic activity dnaE Mutation is lethal 0 b a subunit 3 to 5 exonucleolytic activity proofreading function dnaQ Mutations increase error rate by 103 Initiation of DNA synthesis All DNA polymerases require a primer to provide a free 3 OH end to initiate DNA synthesis Types of priming reactions 1 Primase synthesizes a short RNA primer that is th extended by DNA polymerase cellular DNA papova virus 2 Extension ofthe 3 end ofDNA at anick rolling circle replication oflt1gtX174 3 ProteindNTP primes directly by presenting rst dNTP adenovirus bacteriophage Preexisting cellular RNA mitochondrial genome retrovirus ColE replicon requires along RNA primer ng 12 34 1236 1237 Genes v11 1 Transcription 555 bp RNA primer upstream from origin orreplication passes origin has three hairpins 2 RNase H cleaves RNA primer at originr free 339 OH 3 Persistent 7265 720 RNAADNA hybrid remains 4 DNA synthesis starts replication a RNA primer precursor is a positive regulator b antisense RNA 1103 b is a negative regulator c Rom rotein enhances RNA ImNA primer binding what in ibits rep caljonr transcription continues 6 Mumu39ons in RNA 1 and RNA primer pairing region DNA synthesis is semidiscontinuous and primed by RNA The problem DNA synthesis must always proceed from 5 to 3 As the replication fork moves one of the template strands continuously ex oses new u stream tem l e lagging strand Semidiscontinuous replication leading strand synthesis can proceed continuously in the 5 to 3 direction lagging strand synthesized in the reverse direction as a series of fmgments which are later joined Discontinuous s thesis 5 lagging strand 39 3 Semidiscontinuous replication The lagging strand fragmenm are known as Okazaki fragmenm Usually 1000 to 2000 bases in length GAppp75 3 1 lagging strand 1 2 RNA pn39mer 1112 bases RNA polymeras eqlnaG primase Semidiscontinuous replication The leading strand is also often isolated in fragments due to the misincorporation of UTP Repair of the UTP leaves small gaps until they are filled in pseudoOkazaki fragments lad39 tr d UTP e in San I g gaP 5 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 3 Semidiscontinuous replication Steps in lagging strand synthesis a synthesis of RNA primer b extension of Okazaki fragment DNA 0 synthesis of next Okazaki fragment upstream of the last d removal of RNA primer Who s done it e fill gap and seal ligation nick Semidiscontinuous replication DNA pol I polA starts synthesis at the nick between DNA and RNA and 5 3 exonuclease activity removes the RNA primer and replaces it With DNA 0 DNA polI lagging stmd 2 nick RNA primer 1 5 DNA ligase l LigaseATP complex formed 2 The ATP changes to AMP as is releases pyrophosphate PP and covalently attaches to the phosphate of the 5 end of the DNA 3 Original phosphate at the 5 end covalently joins the OH ofthe 3 end and AMP is released NOTE T4 phagehgzseusesATF E coltligzseusesNAD nicktranslation 5x 3 Ligation Q DNAhgase VPVOVWADorATP O Q 0 T4phagengaseusesATP OVPVO E colt ligase uses NAD 3 70H o 5 Ligase AMP Pr Phosphodiester bond 5 3 o 5 3 3 Ligase seals nicks in DNA 32 muffscams 1 There are two types ofDNA replication in E Cult 1 c1gtX174 phage 2 OriC origin otoactenal cnrornosornal replication There are two types ofDNA replication in E can 1 oxm pnage eacn strand syntnesized separately unidirectional replication fork or Synthesls ofthe r Strand 0 form the doublerstranded elves as a model for laggll lg Strand Synthesls b syntnesis ottne strand to torrn Slngleralrarlda for packaging into pnage particles servers as a del for eading strand sysntnesi ofbaclerlal cnrornosornal replication trands syntnesized at tne sarne tirn bidirectional replication fork 2 ONE origin ootn s DX174 phage as a simple model for replication Rolllng Magma circle 32 replication O O gt 0 gt strand Replicative orm f RF 69 O ds plasrnid strand packaged to form virion Rol ing circle replication is arnodel for lead g s an nt es oxm pnage replication provldes a model forDNA replication RF mndumlymcked ass 0 A REPOWEllEaSE H D Egt E 590 aackground for Rolling circle replication 0 DNA polymerase Juslto separate single strands T 0 kinds of activities are needed to convert double stranded RF DNA to singlestranded DNA without synthesis ofnew DN a Helicase separates the strands iising ATP to provide the energy b singlestrandbinding protein SSB CIDX174 as a simple model for replication Rolling circle replication GEHEA REPA O prdten l Proteins needed for rolling circle replication a gen Aquot proteinto nick at origin pas Covalentlyllnkedto S end oflhe displaced strand b SSB protein to keep DNA singlerslranded Binding is highly coop erativ e c Rep protein provides helicase function d DNA pol 111 holoenzyrne Version 1 Molecular Genetics PCB4522 Spring 2007 Lecture 9 chapter 15 Repair systems section 1520 Repair systems correct damage to DNA Genes viii pp 447463 Types ofdamage 1 single base changes do not interfere wiih transcription or replication examples mlsmcorporatio an match Mi match persists only until next replication one co an ay mutated 2 a1 39 39 structur distortions provide Impediment to puma or transcription examples Lhymidine dim by UV alkylation of G single strand nick removal ofabase Since replica onis impaired must be removed by some om mechanism info next replication 39e M n Mme quotmm 1 Flg 15 34736 Verslon 1 Types ofregajr systems 1 Direct repair Photoreac va on ofpyrimidine dimers rare but is important in plants light activated enzyme p 39 E call 2 Excisionrepair common recognition enzyme see I Hmnn 39 39 39 39 the damaged bases followed by new DNA smthesis Multiple systems in a eell These handle most oftlne repair Types ofregajr systems 3 Mismatch repair mismatches that occur doing replication are eoneeted by distinguishing W 4 Tolerance systems allow replication in case of structural damage accepting high error mte 1m onant in euk otes 5 Retriewl systems recombinationrepair anothertype oftolemnee system Coneets by recombining with a good copy oftlne damaged region Mosdy found in bacteria Major pathways of repair 1 w excisionrepair system 2 dam replication mismatchrepair system 3 7208 and recF recombination and recombinationrepair pathways Version 1 Excisio nrepair systems inE coli Ste 5 n re 1 Mismatch andor distortion of structure exc 2 Incision endonuclease cleavages ofboth sides ofdamaged base 3 Excision 5 3 exonuclease removes DNA between nicks 4 Synthesispo11 5 Ligation ligase seals the nick Version 1 Excisionrepair systems in E coli Uvr system involved in short patch and long patch repair 1 endonuclease uvrA B and C cut DNA at positions 7 nt from 5 side and 34 nt on the 3 side of the damaged base N12 nt total After cleavage this DNA can act as substrate for several systems that excise thymidine dimers including exonl and DNA pol I most important Excisionrepair systems in E coli Uvr system 2 Helicase uer unwinds the cut region to release the strand N12 nt therefore short patch repair 3 99 of repair by uvr system is short patch longpatch repair involves uer protein 4 long patch repair involves removal of N1500 to 9000 nt around replication forks DNA pol l resynthesizes the strand U W system UVrA UvrB recognition uvrA g E 2 DeformedDNA Uvrc UvrB nicking EE 7bp5 amp34bp3 6 helicase amp exonuclease DNA poll uVrD 4 3V DNA poll gt quot9359 5 to 3 exonuclease Excisionrepair systems in E 0011 Mutator mut phenotype Other types of repair systems are high fidelity however Errorprone DNA replication occurs after UV irradiation These mutations were given the name mut Many of the genes originally identified have turned out to be in genes previously identified as components of replication or repair systems Version 1 Excisionrepair systems in E 0011 l The errorprone phenotype may be part of a tolerance pathway not just mutations in repair or replication genes 2 mutations in the umuD and umuC genes abolish induction of UV repair These genes part of an operon that is induced by UV Plasmids carrying homologs of these genes increase UV resistance to killing but the price is increased susceptibility to mutagenesis Excisionrepair systems in E 0011 452 E orprone activity MWLMDC operon encodes proteins that form the UmcD ZC complex UmuD is cleavedby RecA to form D The UmcD ZC complex is called DNA pol V This DNA polymerase can bypass pyrimidine dimers or other bulky adducts Thi polymerase is induced as part ofthe sos system UmuD XZ RecA causes UmuD protein to be cleaved to farm UmuD39 This ls the aeovated farm called DNApul V Controlling the direction of mismatch repair When two normal bases are mismatched how does the cell know which base to change in order to restore the original state replication error Q A Version 1 dam methylation system marks the original strand since it is methylated m A GATC T CTAG replication error Q m A In period before site is methylated new strand is targeted for repair murH mm 063 mum and WVD m GATC indistinguishable CTAG m to repair system dam methylation system marks the original strand since it is methylated m GATC o CTAG replication error Q m E can dam39 strains A show high rate of spontaneous mutations O W my mum These are mutations 0 MMS and WVD during replication Q n A GATC o T CTAG Newly synthesify m DNA MutS dam methylation mismatch G q MU re air QQ p a MutS MutS translocates to GATC site Nonrnethylated strand is back to the damaged site as an endonuclease to New strand nick the uer unwinds the synthesized by unmethylated strand nicked strand DNA pol Version 1 Default repair systems that show bias in error correction c lemma w T VSP system uses MutSL to remove T from G T G C GT and CT mismatched I I I I pairs Not dependent on GATC site MutY removes A from A and GA mismatched Does not use the n glycosylase which cre apurinic site n o n o a n 9 s 2 mm o So39 m S E 8 E E E lt Retrieval systems in E 001139 Also known as postreplication repair or recombinationrepair ll damage remains T 5 T singlestrand exchange Q 4 4 3 retrieval Version 1 Retrieval systems in E 001139 Role of the recA protein 1 In uvr excision repair mutants introduction of a mutation in recA eliminates all remaining repair 2 Replication in uvr 39recA 39 double mutants resulw in production of DNA fragments Whose size corresponds to the distance between thymidine dimers More than 12 thymidine dimers are lethal Wild type can tolerate up to 50 Retrieval systems in E 001139 Activities of the recA protein 1 The recA protein is involved in normal recombination and in singlestmnd exchange of recombinationrepair 2 The recA protein is activated by UV irradiation and is thought to induce latent rotease activi 39 is target proteins LexA repressor A repressor An SOS system of many genes Stress conditions that damage DNA or inhibit replication induce a family of genes that comprise e SOS e ponse All ofthese stresses initially act upon the recA protein Which in turn inactivates the LexA repressor Induction conditions UV crosslinking and alkylating agents thymine shortage and mutations in some dna genes singlestranded DNA and ATP39 same as required for RecA function in recombination The SOS system repair system includes a bypass system tolerance system that allows DNA replication across damaged areas at the cost of delity SOS repair is a ma39or cause ofUV induced mutagen 1s The E rotein ibit the editing function ofDNA pol III by directly binding to the thymidine dimer Mismatches in the opposing strand are not corrected Verslon l An SOS system of many genes su ll amy egarding the mechanism ofch ste r activation Inducer may be an intermediate in D N small molecule released from DNA singlestranded DNA and ATP 7 these are suf cient m mm m RecA activation of LexA lexA actuated recA activated recA 7 quot1133 O Q we gt gt O O 3 00 8 lexArepressnr O inductinn gt gt o gt sosm repmrgene O repmrgene Regulation of uvrB by dual promoters 0 lexArepressur recA gene 1m gene meuterrl meuterrZ repressed by lexA cunsmuuve Version 1 All targets ufrecA are cleaved atthe d Activation of lexA Q Q Gly Very hm ammu acids in leXA suggesting that pmtem 0 structure plays an lmpunant lgt rule in target recugnmun pends Ale e urnu Ether eemmer alareg Regulation ofthe 0 90 SOS system amp nurmally keeps levels uf RecA and EXElSan luw XA rep air enzymes repalr g cm lexA g Q Cg Q RecA gene byrprnducts nf UV DNA damage QM 0 Q 0 repairgenes lexA O LexAxsalsnmduced O butlsrapxdly 00 gt gt o gt O mamvated by RecA lexA O O G O O RecA gene LexA gene 0 Vers on 1 A er damage is repaired recA l5 nn lnnger amvated Tms results m the O0 Equot budldeup nf LexA pmtem which shuts 00 C dde the sos system tepaat genes cod ex 0 1 A0 Ge a O RecA gene 00 LexA gene Mammalian repair systems Characteristics 1 only 34 bases replaced shortpatchrepair only 2 MSH tepaat system m yeast is homologous t e E call mu system This system tepaats mismatched base paats Mammalian repair systems m 3 human hereditary disorder xeroderma pigmentosum XP resulw in extreme sensitivity to sunlight Explained in terms of a failure in excision repair of 39midine dimers Nine complementation groups characterized by de ciency in excision repair Version 1 Mammalian repair systems Chamcteristics 4 In yeast the RAD gene producw RAD3 RAD6 amp RADSZ are involved in repair of radiation damage UV and others RAD3 is involved in excision repair One of the genes responsible for XP is in the RAD3 group ofhumans RAD3 is ahelicase subunit of transcription factor IIH T FIIH Note than in eukaryo es N repair is closely linked to transcription A common system repairs doublestrand breaks Genes VIII section 1529 No des but read the text and understandthis
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