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by: Cameron Koss I


Marketplace > University of Florida > Biology > PCB 5530 > PLANT MOL BIO GENOMIC
Cameron Koss I
GPA 3.73

Gary Peter

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Gary Peter
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Date Created: 09/18/15
Plant Molecular and Cellular Biology Lecture 2 Mechanisms of DNA Repair Gary Peter Figure 5 51 Molecular Biology ofthe Cell 4th Edition 8202008 332 Learning Objectives an 9 mmatzjd strand 1 Explain the role for w I DNA repair in high fidelity DNA replication amazes 2 Describe the structures and functions of e n n S i b I e Fire 5 49 part 1 of 2 Molecular Biology of the Cell 4th Edition for DNA repair 3 Explain their roles in DNA repair 4 Explain the in vitro applications of DNA repair enzymes for recombinant DNA 820200m PMCB Lecture4 G Peter J Mutations and DNA Repair 5 o Mutations occur if the DNA is not repaired o The damage that occurs is usually to onetwo bases on one strand thus the opposite strand provides the template for insertion of the correct base deaminatediC39 f new strand a g has been changed39toa39n7A DNA REPLICATION new strand old strand A unchanged Figure 5749 part 1 of 2 Molecular Biology of the Cell 4th Edition mutated ol strand new strand an 39A l39gnuc leotid39e pair hias be39en deleted Bl 8202008 PMCB Lecture 4 3 Peter Figure 5 49 part 2 of 2 Molecular Biology of the Cell 4th Edition 3 O O O O I I 0 O O O O f DNA R VGI39VleW O epalr o O O O O O spontaneous reactions oxygen radicals UV light alkylating atkvtating agents enwrormental replication agents ill rays mutagen s Xraya Errors OSmeG abasic suites pyrimidine dimers doublestrand base msmatches midi ed dea mlina ted bulky addu cts breaks DS Es insertions atkylaied bases deletions ssDNA bre alts l a ti s it direct repair 3 39ui 39 Vquot 39 L 39 a mismatch repair repair BER repairWEH recombination HR MMFtl nonhomologu us end Joining MH EJJ o DNA repair is essential for survival o 5000 purine bases are lost per cell per day from thermal fluctuations o Increases fidelity of DNA replication by 100 fold over DNA polymerases 8202008 PMCB Lecture 4 3 Peter 4 39 39 O O O O ommon eps In epalr on O O O O O O O O Pathwa s O O 0 Recognition of the altered base Facilitated by structure of double helix 0 Removal of alteration 0 Synthesis of correct nucleotide o Facilitated by the structure of double helix amp sister chromatids 0 Some exceptions occur here direct reversal nonhomologous end Jornlng error prone DNA repair spontaneous reactions oxygen radicals UV ligh1 alkylatlngr alkylating agents enwrormerltal replcaron agents Xrays mutagens Xrays anors OSmeG abastc sites pyrimidine dimers doublestrand base msmatches nxidimd deaminared bulky adducts hreaksl DSBs insertions amylaer bases deletions ssDNAbreaks I 1 1 3 3 direct repair baseAexcision nucleotideexcision homologous mismatch repair repair BER repairl39NElW recur rnbinatinra tHFljl MR nonhomologous 5 82 and joining NHEJJ Common Forms of Damage to Bases in DNA Figure Ha Molecular Bialugy elm Cell JlIxEdnlan DEEU JNATION l U H 4 NATH H ll GUANINE DEAMINATION H30 NH DNA Figure 547 pan 1 or 239 Mnletular Biology loe Cell Am Edition 0 The glycosidic bond of nucleotides is prone to acidcatalyzed hydrolysis to form abasic sites 0 Detection and repair of damaged bases 10000 abasic sites per human cell per day 100003x109 1 in 3 x 105 bases is facilitated by the structure of DNA 8202008 PMCB Lecture 4 3 Peter O O O O C O O O O O O O O O O C 39NA IZUBALEDNAiEASESE WM um I l ll bug39s3555 H20 0 H N 4 N ELAN H H lt71 NAM NAH NI l adenine hypoxanthlne 0 H20 0 H H N N ll 39N N V N ill W1 H Quanlne xanthlne H H20 0 H H H flu l N 0 gt J H N H N O lll cytosine uracil O ch l N W as I 0 NOjD EAMlNATlOM H N th mine A y ll H H20 0 H C H3C H 3 N 1T H NAG H NAG l NH l 5methyl cytosine thymine 15 Figure e52 part 2 er 2 Molecular Biology ofthe Cell 4th Edition Direct Reversal a Two types of altered bases can be directly reversed V 8202008 alkylating OGalkylguanine adducts are directly removed by O6 alkylguanine transferases Pyrimidine dimers cinema induced by UV light are directly reversed by photolyases PMCB Lecture 4 3 Peter UV lig hl enwrormental mulagens pyrimidine dimers buier adduc rs 3 nucleotideexcision repairiNElii l Direct Reversal of Damaged Bases OGalkylguanine Transferase g c I J o In most organisms O6 alkylguanine adducts can be repaired by alkylguanine transferases AGT o AGTs transferthe alkyl group from the altered fka Fi gm guanine base in the 02 O The EMBO Journal Vol 19 pp 17191730 2000 N N N NH dsDNA to a reactive s lt I 54 I llKN 0 82 I M JNH2 t DNA DNA cys eIne group In an 19 m ltN NJ NH H irreverSIble reaction Scheme 4 Mechanisms of repair by Taalkylguanine transferase ACT ACT restores a guanine resi us in DNA by transferring the meth I group from wmethylguanine 15 to a reactive cysteine residue ofthe pru ein in an 39 reversib e rea ion ooBenzylguanine 21 is a potent inhibitor oFAGT that is in clinical development Fur antitumor therapy 8202008 PMCB Lecture 4 G Peter 8 Angew Chem Int Ed 2003 42 2946 2974 Direct Reversal of Damaged Bases Photolyases o UVinduced pyrimidine dimers cyclobutane pyrimidine dimers can be repaired by nucleotide excision mammals or by direct reversal of the damage by photolyases in plants lower eukaryotes and bacteria Photolyases contain a chromophore which absorbs light and transfer of the excitation energy to the FAD cofator for electron transfer to the pyrimidine dimer to initiate the reversion Current Opinion in Chemical Biology 2001 5491 498 Base Excision Repair DNA Glycosylases o Damage to DNA bases from deamination oxidation demethylation and alkylation is mainly repaired by baseexcision repair 0 Multiple different DNA glycosylases recognize specific damaged bases and initiate their repair by base excision o Repair is then subsequently completed by a number of different enzymes 8202008 O C C O O O O O O O O C spanianeous reactions oxygen radimls alkylating agents Kways abasic sates nxsmmd deaminaled alkylarad bases sle lA breaks 5 baseexcision repair BER Tabla 1 Human DNA glycosylases Enzyme Most important substrate AP Iyase UNG U SOHvU in ssfdsDNA no SMUG39I U SOHU in ssfdsDNA no TDG UG T16 t39C no M804 UG TIC no 0661 S oxoczC fapy yes MYH AzgoxoG no NTHl oxi pyrimidine Fapy yes NEI39I ox pyrimidine fzipy yes MG MPG 3MeA TMeG 5A Hx no 10 PMCB Lecture 4 3 Peter AngeW Chem Int Ed 2003 42 2946 2974 DNA Glycosylase Uracil 53 Glycosylase Deaminated cytosine residues are uracils Uracil DNA glycosylase recognizes the UG mismatch and excises the base from the DNA strand by the hydrolysis of the Nglycosidic bond between the base and the sugar phosphate leaving the backbone intact and producing an abasic site AP endonuclease hydrolyzes the phosphodiester bond 5 to the abasic site to generate a nick Removal of the abasic site occurs by the AP lyase activity of DNA polymerase B DNA polymerase 3 also adds the single new C and DNA ligase seals the nick A BASE EXCISION REPAIR deaminated C 5 G C T U A T C C hydrogen bonded base pairs CGAGTAGG U R ACIL DNA U GLYcovaASE DNA helix with missing base AP ENDQNUQLEASEAN D RHQSPHQDIE39STE R555 a REMOVESUGAR PHOSPHATE DNA helix with single nucleotide gap Hquot i quot39l quoti CGAGTAGG 7 7V 4 pNAPQLYMEBASEADDSNE W I NUCLEOTLQES39 DNA LlGASE SEAL SiNlCZK39 G C f f Figure 5 50 part 1 of2 Molecular Biology of the Cell 8202008 PMCB Lecture 4 3 Peter Uracil DNA Glycosylase Uracil DNA glycosylase is the most active of the four DNA glycosylases that show activity towards deaminated C uracil o 105 faster than other DNA glycosylases UDG interacts with the replication machinery and appears to clear the genome of U immediately after replication All DNA glycosylases use a common nucleotide flipping mechanism o Target nucleotide is extruded out of the dsDNA into the active site UDG and SMUG1 are the only glycosylases that act on both ssDNA and dsDNA 8202008 PMCB Lecture 4 3 Peter 12 AngeW Chem Int Ed 2003 42 2946 2974 In Vitro Use of UDG 3 o Preventing carryover contamination in PCR 0 Incorporate dUTP by Taq into PCR products o Treat with UDG to produce abasic DNA which will no longer amplify 8202008 PMCB Lecture 4 G Peter 13 I I I I Nucleotide EchSIon Repair 33 o NER is the repair pathway that removes a variety of bulky adducts from DNA 00 o Pyrimidine dimers UV 0 Environmental mutagens ll39u39l39h39 o Chemotheraputlc agents 00000rfJanal mutagens 0 Two maIn pathways 0 Global genome repair GGR for untranscribed regions the bulk of the genome o Transcriptioncoupled repair TCR pyrimidine dimers o This broad substrate specificity is remarkable when 00100000000 compared with the specificity in base excision repair 0 The efficiency of repair of the different adducts can ll vary several orders of magnitude and generally correlates with the degree of helical distortion caused by the alteration 0 Note simple mismatches or bubbles are not 0202000 substrates so distortionrefetherNA backbone is 14 insufficient Nucleotide Excision Repair E coli 0 This pathway requires the uvrABC encoded exinuclease a helicase encoded by uer and DNA polymerase 8202008 UvrA is both an ATPase and a DNA binding protein it contains Zn nger motifs It functions as a dimer and it recognizes and binds to damaged DNA The function of UvrA is to lead UvrB to the site of damage UvrB is an endonuclease and an ATPase although the ATPase activity is cryptic and is only revealed when it is complexed with UvrA UvrC then binds to UvrB This complex nicks the DNA on either side ofthe lesion or damage UvrC nicks DNA about 7 nucleotides on the 539 side ofthe damage UvrB nicks DNA about 4 nucleotides on the 339 side ofthe damage The Uer helicase binds to this region and unwinds it By so doing it displaces the short single strand carrying the site of the damage In total a region of 1213 nucleotides is removed This region is then repaired by DNA polymerase I and DNA ligase PMCB Lecture 4 3 Peter 1 1111111111 7 13977111111111 3 lllllllll tan u Excision of a IZnurleolida fragment by uvrABC excinuclease 1111 DNA synthesis by DNA polymerase I MW l lllllulillll 111i111111111 Joining by DNA llgase lll111ll Illllllllllll Mechanism of NER GGR in Eukaryotes recognition 0 The steps are a IHHHJUIHHHCIIIII lllllllllll 5 TFIIH binding and 0 Recognition of damaged pammmmg residues Bubble formation Dual excision of the WEWW G u l damage verification damaged DNA strand 5 and 3 to the lesion o Release of the 2432 bp oligonucleotide containing the damage Repair synthesis Ligation of the gap 1 dual incislon repair synlhesls i I a ligation 39 8202008 PMCBLecture4GPetel 39 quot quoti quot quotHHI AngeW Chem Int Ed 2003 42 2946 2974 l 1 I o The core NER reaction has been reconstituted in vitro and about 30 proteins contribute to NER o Damage recognition amp bubble Mechanism of Global Genome 555 Repair 25 l39iill ill Ilnll IlilllllIllltl39ll initial damage recognition i l illtll ll ldlllllli lllllllllll f0 rmatio n b TFIIH binding and padlal opening 0 XPChHR23B TFIIH 9 subunits includes helicase XPA RPA trimeric SSB XPG Incision amp release of oligonucleotide o Endonucleases XPG and ERCC1XPF DNA fillin o Pol 5 and pol a sliding clamp PCNA pentameric clamp loader RFC In full helix opening damage verification Ligation a Emma39s BQOOOOEDNA Iigase I PMCBLecture4 3 Peter 7W ml m m III39HI39II 7 AngeW Chem Int Ed 2003 42 2946 2974 Mechanism of Transcription coupled Repair 0 TCR was discovered from observations that lesions mm which block RNA polymerases are repaired more rapidly in transcribed parts of the genome Initial damage is recognized by stalled RNA polymerase All the proteins involved in GGR except XPChHRZBB are required for TCR TCR requires additional proteins including CSA amp CSB Assessment of the obstruction by TFIIH XPG if ultraviolet photoproduct I If oxidized base recruit speci c recrurt XPA and other factors tor NER glycosylase and other laclors for BEFl 8202008 PMCB Lecture 4 3 Peter 18 Mismatch Repair o The MMR system increases the fidelity of DNA replication o Eliminates base mismatches nucleotide deletions and insertions introduced by DNA polymerases o MMR enzymes recognize 1 2 chemically altered bases in a mispair or loop 0 MMR is conserved from bacteria to humans but with some notable differences o In prokaryotes the newly synthesized DNA strand is recognized before it is methylated o MMR enzymes recognize the hemimethylated DNA and act only on the unmethylated strand Eukaryotes don t have hemimethylated DNA after DNA replication and the recognition mechanism is unknown 0 One model shows that the MMR machinery is coupled to the replication apparatus 8202008 PMCB Lecture 4 G Peter 19 8202008 Mismatch Repair E coli In E coli the initiator is the MutS homodimer which binds to mismatches and insertiondeletion loops 0 Binding of MutS to the alteration is the rate limiting step for repair Upon binding MutS triggers ATP dependent assembly of the repairosome during which MutS moves away from the mismatch and MutL homodimer is recruited MutL acts as a bridging factor for MutH which nicks the newly synthesized strand 5 of the nonmethylated GATCGAmeTC This nick serves as an entry point for helicase II and one of a few exonucleases Exo VII Rec J or Exo l which degrade the nicked strand past the mismatch The ssDNA is protected by 888 The gap is filled in by DNA pol lll DNA ligase repairs the gap 239 error In niele BINDING OF MlSlVIATCll made strand PROOFREADING PROTEINS NA SCANNING DETECTS D MutS MutL NICK IN NEW DNA STRAND STRAND REMOVAL REPAIR DNA SYNTHESIS Al Figure 5 23 Molecular Biology of the Cell 4th Edlti PMCB Lecture 4 G Peter 20 Structure of E coli MutS 55339 o The two identical subunits interact with the mismatch as a functional dimer o One interacts with the mismatch o One interacts with the parent DNA o DNA is kinked by 60 at the site of the mismatch 8202008 PMCB Lecture 4 G Peter 21 Angew Chem Int Ed 2003 42 2946 2974 Mismatch Repair 0 No mutH similar genes are in eukaryotes consistent with a different strand recognition mechanism o The MSH6MSH2 MutSoc heterodimer recognizes the mismatch to initiate repair o MutSoc binds to single mismatches and to smallinsertions and deletion loops o MutSB only binds to insertiondeletion loops of various sizes 0 ATP hydrolysis drives the threading of the DNA through the MutSoc o MutSoc or MutSB dimers trigger assembly of the MMR machinery o MLH lPMSZ MutLoc and PCNA are recruited o PCNA is the processivity factor in replication 0 Exo 1 is involved with MMR but 82079ng other exonucleases or helicases have been identified PMCB Lecture 4 G Peter Eukaryotes zen G T masmmsnz J 3953 9 5y 3 MLH1IPMS2 PCNA ATP AJJP 5393 exunuclasa DNA helicase 39 G UNA P la RFC PCNA RPA l DNA 9 G C 3 Angew Chem Int Ed 2003 42 2946 2974 7 Double Stranded Breaks 55 0 Double stranded breaks are induced by ionizing radiation oxidizing agents replication errors and specific metabolic products cells 0 Unrepaired lesions would quickly lead to the breakdown of chromosomes into smaller fragments ACCIDENTAL lBREAK LOSS OF NUCLEOTIDES DUE TO DEGRADATION FROM ENDS 8202008 PMCB Lecture 4 3 Peter 23 Repair of Double Stranded Breaks Two independent pathways 0 Homologous endjoining 0 General recombination mechanisms transfer the sequence information from the intact chromosome to the site of the dsbreak o This HR pathway is important for meiosis repair of interstrand crosslinks o The HR pathway is important in S and G2 phases during which the sister chromatid is available 0 Nonhomologous endjoining 0 Broken ends are juxtaposed and rejoined by DNA ligation with the loss of 12 nucleotides at the ends 0 NHEJ pathway appears important for quiescent or terminally differentiated cells and in G1 0 NHEJ is required for immune diversity and telomere maintenance 8202008 ACCIDENTAL lBREAK LOSS or NUCLEOTIDES DUE TO DEGRADATION FROM ENDS COPYING PROCESS INVOLVING HOMOLOGOUS RECOMBINATION g1 complete sequence restored by copying from second chromosome 3 HOMoLoG39ous39ENDJQINING 4 region with altered segment due to missing nucleotides IAI NQNHQMQLQGOUS END JOINING Figure 5 53 Molecular Biology of the Cell 4th Edition PMCB Lecture 4 G Peter 24 Repair of DSBs by Homologous Repair 0 HR is a highly complex pathway 8202008 End recognition and processing 0 Processing the ends of the break by RAD52 and exonucleases generate 3 single stranded tails PMCB Lecture 4 3 Peter Angew Chem Int Ed 2003 42 2946 2974 O O O O 0 O O O O O O O O O O O O Q I 5 a USES 5 a Fiad52739 a 9quot 5mm HadsONJre11JBbst and pmoesslna nuclmcla processed ends EPA H3651 Rad52 b strand Invasion Rad54 Rad51 pamlngs Bmaz joint molecules a repair synthesis Poiymerase ei branch migration 4 modeled lemurs Hulliday iunctiuns d branCh gram Holliday junction resolution resolvase t Mus 31 recombinant molecules Strand Invasion Homologous Pairing and Strand Exchange 5 0 Strand invasion 5quot 8202008 RPA binds to the 3 overhangs Rad51 a recA homologue assembles onto the single strands to form nucleoprotein filaments Nucleoprotein filaments search for homology in the donor template 0 Rad54 facilitates this process by interacting with Rad51 and stimulates the strand exchange reaction 0 Rad54 is a Swi2Snf2 family member involved with chromatin remodeling HadEZ Flad51 paralogs a Flede Y PMCB Lecture 4 3 Peter Repair Synthesis and Branch 5355 I I 0 Migration a 0 Repair synthesis is clzismszz ifgggmm mediated by DNA Em polymerases 0 In E coli RuvA and i aliasing RuvB proteins mediate branch migration and Rqu catalyzes resolution of Holliday junc ons o RuvARuvB form an active complex 8202008 PMCB Lecture 4 G Peter 27 Angew Chem Int Ed 2003 42 2946 2974 Nonhomologous End Joining o Ku70Ku80 heterodimer binds to the ends at the endmng i 39 dsbreak and recruits DNAPK DNA protein kinase 0 Bridging of the two ends requires the Rad50Mre11Nba1 and Lig4XRCC4 complexes which both interact with the Ku proteins 8202008 PMCB Lecture 4 3 Peter 28 DNA End Recognition by Ku70l80 o Ku70 amp Ku80 share limited sequence identity but show similar folding patterns 0 They bind to the DNA as a dimer 0 DNA binds through the ring and the Ku proteins do not directly contact the bases only the phosphate backbone 8202008 PMCB Lecture 4 G Peter 29 Inducible DNA Repair Systems 53 SOS Response in E coli m o The 808 repair system is induced in response to major damage to the bacterial DNA or in response to agents which inhibit DNA replication 0 The system is a complex one with over 20 genes involved Two of these are the important regulator genes lexA and recA o LexA is a repressorthat regulates the expression of all of the other 808 repair genes including recA It also regulates its own synthesis ie it is autoregulatory Normally LexA blocks expression of the SOS repair genes 0 The RecA protein is a multifunctional protein with ATPase and ssDNA binding activities When bound by ssDNA it is also a co protease Damage or severe stress to the cell generates ssDNA which activates this coprotease activity The RecA coprotease activity upon binding to LexA stimulates the coprotease activity of the LexA protein As a result LexA is no longer able to block transcription and the SOS repair genes are thereby induced and expressed 8202008 PMCB Lecture 4 G Peter 30 Inducible DNA Repair Systems 53 SOS Response in E coli m 0 Among the genes that are induced are uvrABC and and also umuC and umuD UmuD is cleaved by the RecA coprotease activity and the truncated protein UmuD39 in association with UmuC forms DNA polymerase V Pol V requires the B and y subunit of Pol III for optimal activity DNA synthesis by Pol V is error prone Error prone DNA synthesis can be harmful to individual cells but must be advantageous for the population 8202008 PMCB Lecture 4 G Peter 31 Summary 3 0 Multiple repair systems have evolved to protect the genome from mutation 0 These systems involve multiple proteins with multiple different functions a Each of these systems must identify the few bases that need to be repaired in the genome 8202008 PMCB Lecture 4 G Peter 32 Plant Molecular and Cellular Biology Lecture 2 Fidelity of DNA n m m t e w P m N e a R G 666 7239 246 2 2 k 2 7239 8239 A 8239 AV 8239 ZP K8239 8239 REPLICATION EPLICATlON REPLICATION Z2 1A Figure i5 Molecular Biology of the Cell 4th Edition 1 Learning Objectives List and explain the mechanisms that insure highfidelity DNA replication 5 Explain why the fidelity of 39 DNA replication is so important for survival and for recombinant DNA work newly synthesized leading strand parental newly synthesized lagging e 39X strand C Figure 5 22 part 2 of 2 Molecular Biology of the Cell 4th Edition Importance of HighFidelity 5 DNA Replication o If1 mutation occurs per 109 bp 0 Then what39s the mutation rate of a 1000 bp gene in a population of 106 bacteria 0 What s the frequency of the mutant in the population 0 0 0 0 Generation 1 o 106 copies of 1000 bp gene or 109 bp to be copied 1 new mutant 2 x106 ces 5x107 Frequency in pop12x1065x10397 Generation 2 0 2x106 copies of 1000 bp gene or 2x109 bp to be copied 2 new mutants 4x106 ces 5x107 Frequency in pop44x1061x10396 Generation 3 0 4x106 copies of 1000 bp gene or 4x109 bp to be copied 4 new mutants 8x106 ces 5x107 Frequency in pop128X10615X10396 Mutation rate is constant at 5x10397 Frequency of mutants in the population increases Consequences of High Mutation Rates 0 Mutator strain propagated for various cycles 40 cycles 1000 generations TABLE 1 Phenntypic tests for loss of gene functiun 39l39t39 Ls No If go nm mxmrnnlw lilnlng l hr lullllly i5 MINE In rl l39 Sugn r l39re rnmn l u non Phage I CESISIEJIIEI Spaclnllzml Ell Cont llllnnal Irsllml 4 l EDI 35H il All 5 3 5 5 5 1 Ll 39l quul39 l The lnrrunst In rlmlnllnm ll ml C 50 loss of germ l39ImElmn quotmung I lln mp 110 real Ills EII39K IJIOIIMJ l39nr Llll39l39rsrrenl Is 3 s I39llr l39l l lrclvs gt sum IlllllilllEJlls llml ilthE one of Ilw hll ll IIIlllmllun pathways 39lht39v sulld ctlltzlns t Irp It sonl ll1llFJIIEZIl12l rmsnlllnpI In 11 Imlmplu39 Illt39 SEJIIII mlllnrm I rqurtsonl nu lIaIIans C39JI Islng Inn39s 3f mullllly llm solld lrlanglrs Ill mpm ernl nmlnllom Elt rlll r39cl lIy III 1th pln Im cm I lug loss of L 39 lIlll IIID nptsn ll39llllquotl5 I mpresenl rnulznluns msulnng In I39llgh Hall sonslllvl 1339 3 H 7 S Funchain et al 2000 Genetics 154 959970 Figure 4 5 Molecular Biology of the Cell 4th Edition What Processes and Pathways are Important for DNA Replication o Monomer biosynthesis 0 De novo and salvage pathways for nucleotide biosynthesis o Purine and pyrimidine biosynthesis o Nucleoside monophosphate conversion to triphosphate o Polymer biosynthesis Template Primer a DNA polymerases o Other enzymes Method for Measuring the in Vitr Fidelity of DNA Polymerases 0 Why use M13 o What does the host need 0 Can this assay be used to measure the fidelity of all polymerases o What do you expect the results to be r a I v In Vino DNA Inmlmrr I H n a huhquot O O w W Milling Edmund FINA FlaIi ATGC Furlfr DNA Seau Mullfln 4 and SliuIHl Fro41er ind miimm uum NitricIran MJrunlillith bluI 9r calBrien FIG 1 Experimental outline of MILEmull mnmg nm M 511339 Tim rm pairs of 41an an the gripped molecule indicate the position oi the ve sites for Benin mathylntinn llii d In tumum mismatch annualjun E20 3923 The gap extends Illum positicm 174 to 216 where 1 is the start of twnac miun nnd is lkamrminull hry clmwngr with mmuhm endunurlmaes PD l and PL39H39L The 5 and on the left of the Wham gap is therefore mum than 1011 Manes away mm lhc and n Lire trgei which in position lrl the rst nuclemjrle alter the lad gene texminmion radon but will within the rag DNA Tm Ll H primer tentmus on the right is nurleoti 113 the middle mIELECIUd pf the haze sedan 45 The mm tramL is indicated by the 1151er Jim Within the gap The dimLinn Of DNA synthesix within the gap is right m left The 317er lml k With dashEl lines represent a competent null null Kunkel 1985 JBC 260 9157875796 Results of Assay O O O O O O C 0 TABLE ll Mumzim fWurrm39 of mnlml and m nJianm39rd pm 0 C The rat hepatuma DNA polymerase copying reactions tramch tIDHS and milling were as clawide under Expe mental Proceduresquot and in Ref 17 Mum mt I rcquencies a viral and replicatlve turn DNA WEI d mrmined Item the name pmurntinns of DNA used 113 mnmmcl LhEr gnpped molecule N Ndurlibca39crf Fianna1 acmd mm Tami ll 1 A nlmnu v nalium Tm Muzm 1mmquot Mutation fnmncncyaf Dim mpwd by PailIll fmm dr arcnr WHW39E H 39 7 7 739 V 39 xiv fuming reacllcma transfect39mm and plating were 85 l JJE BHib l Erica 2 10597 739 LE under quotEmerimantul chnclu39res HEpll lwe funn 2 231 B 543 39 39 f quot r3 7 It commaquot u wanna 123 14 Suunzc or Number mlquot 4quot 91quotquot Mmtinn u caaq391i39m39 5 10474 68 64M Purl1 dct minntitms mmmncy l al miecl dsmwred39 r 1 2109 i 9 Ac 7 139 quot 7 7 7 quot Mumms include calmless plaq lwa an wwll as than having light Em blur calm lib1n wild mac mp2 Sewml lighl blue phemtype39s were Hat hepnmma 3 lug Til SEE Milli nimnml varying in immensity fmm almost micaless to almost wild Chick emh 4 ELEM l illELL 11119 Marl than 95 9f the mumnm when carefully removed from H L n 39 1 4 415 161 TE n lhB Dim dilutes and ruplated mm of only a single phonmypc e E m V quot a 2 39 Occasionally a plaque havng animus le In lh calming and blue H 2 phenqylypes was gingerwed which when updated yielded bath wild type 7 ll IE plaques and mutant plaqucs in nppmximnm pmparticm m the sinc ol39 the mewm in we nriglnal infective center The ratio ul39 Light blue to calmlean mutants was 21 for the nirkucl muntrim transfer tirins hut 12 Tar Line Pollicopied DNA This is consistent with the sequmce analysis Table Iquot since many of tin I al framesth mumnls pmdmzecl a high imminamzjww an C l l lE S quot The nicked camtmcl was made her clawing plinthm form DNA with rLstritrnicm untflunuclcau Ann and linen hybridizing the full genome length clumplememary strand to the viral strand us far foxmania ml the garland mullsink The mauillng mmpletely double stmnded circular molecule mncajm a nick lit punitian 2 d unly l bases mm the pjaininn if the nick in the Ful gap lled molecule Having laser aubjmzlecl m manipulatium imilar m the gapp l mull cule and having a similar cun gmaunn La numpIc39lely double simeled circular with a single nick outside the mututiunai Inrgnl hut wilhin the nunessential DNAL lth cumtruchnn was themed most pmUpdate lur sulassequcm analyses of smnianmua mutant Table nu Kunkel 1985 JBC 260 9157875796 Frame shifts and substitutions are similar in frequency ThBILIF III qugnqlofjn lw Elma nil Inuktitut 393 Spunmnmu Rat I Mi3 Elmk Pali Mumnmml my Hm I Miquot H40 39x m quot3 mm x ur39i V Number fteqqu39 3 er g 7 7 7 xm xiii4 7 7 m7 iflngle lh lse Frame shif l 11 055 34 355 50 12 minglehm sul aimtmn I37 34 1392 220 7839 190 Delainng 32 LG H 24 13 32 n her 13 LEI 9 41 3 139 H 39l39qu 191quot 64 279639 54th 144 am SWle J jtEiu mutants in mm Lrumt scliun cf the nicked canamcl DNA issue Tabl l and and nut Mcmmriiy ladene indent 39 The muijan requency f0 unnsfvrtion of chuck PolHwpled DNA in ICIWN39 here than in Tnlnle ll pranunwhly dun w 11 decde modulation if 391 frame u hif ls resulting from H Eik39nt T C Chung ul ponili xn 72 ls EMMA 39 op simpli camm he mJuns in this column are detzrmimxl using a total forward mutation fmerlfy ul l x mquot obtained using DNA cnnlaining a T residm st Fusillun 72 Since 137 of 236 mutants were actually obtained using a wrgm mmuiulng a C maiduc at mailinn 72 the cure null lme mutation frmumn39 for with Class of mutations is actually aligluly different than the values shown The Effect is small and docs nuL albur lln conclusions Other ilnclud u lupliunlimm dauble mutants armpitx mummm 6139 several types and for the atammxmm collection ll muu nu exhi ing only a very slight reclmzljnn in blue rail intensity and for which ncl Change has n mum from pn iliunx 54 tuxmug 134 L he Hiram within the gap Two of the thug ut hgr mulnliuns I ur the chick Pnl caiinctinn were also at this type Tho Pol duuhle mumu39om mmajn two widely swarmed mumtiunai events in the same mlaleizulv Jmllmallljl Mleien ng tum inLlerJEnLl nl an urs by base inaccurate enzyme Kunkel 1985 JBC 260 9157875796 Why M13 55quot o Facilitates creation of gapped duplex o Non essential gene and occomplementation already present o Large dsDNA region contains adenine methylation which should help limit the mismatch repair of the mutations arising during in vitro synthesis o Easy to score large numbers of plaques Single stranded phage are readily sequenced What does the host strain need o The rest of the lac Z coding sequence for the intracistronic complementation to work o This is located on the F plasmid to insure that susceptibility to infection by M13 phage Can this assay be used to measure the fidelity of all polymerases 39 only those 5 Si polymerases that quotquotiiquot quot can use gapped met templates 0 Template 0 Primer K Fidelity of Various gigs Thermostable DNA Polymerases 55339 DNA Polymerase Error Rate x 10396 Accuracy x 105 Mutation rate per bp Error rate1 Pfu 13 02 77 Deep Vent 27 02 37 Tli VentR 28 09 36 Taq 80 39 13 UITma 553 20 02 Accuracy is the average number of bases duplicated before an error is made 0 What does this mean for your recombinant DNA work The Impact of Error Rate on of clones with sequence errors o Error rate of PquItra is 4x106 and Phusion is 14x106 o The number of mutations per bp equals the error ratenumber of doublings o Mutation frequency is MFERbpdoublings mm 1 kb 07 24 5 kb 34 119 10 kb 68 238 Impact of Reverse Transcriptas 5 Errors on cDNA Sequence EE 0 AccuScript RT 0 ER 16x 10395 0 So for a 1 kb cDNA expect 16 of the clones to have an error o After 20 doublings 106 amplification expect Error Rate 106 Mutant Clones Pqultra 043 25 PiCOMaXX 40 96 What s the Normal Mutation 5 Rate 9 o The probability of a mutation occurring due to random chemical decomposition of nucleotides is estimated at 110000 bp 0 The standard fidelity of E coli DNA replication is 1 error in 1 billion base pairs REPLICATION STEP ERRORS PER NUCLEOTIDE P OLYMERIZED 53993 polymerization l X 105 339 gt539 exonueleolytie proofreading i x IDg Strantl clireeted mismatch repair i x it2 Total t x 103 The third step Strant i direeted mismatch repair is described later in this chapter Four Mechanisms that Control the Fidelity of DNA Replication E o Complementary base pairing 0 Conformational change of polymerase which delays addition to a growing chain allowing additional time for the incorrect nucleotide to dissociate 0 Removal of incorrect nucleotides at the 3 terminus of the growing chain by the 3 to 5 exonuclease function of the DNA polymerase 0 Removal of mispaired nucleotides by the mismatch repair system Summary 9 o The fidelity of DNA replication is due to the structure of DNA the 3 5 proofreading activity of DNA polymerases and DNA repair enzymes 0 DNA replication requires the concerted activity of multiple enzymes which bind together at the replication fork into the repliosome Nucleotide Levels in Cells 555539 9 dNMP 43 FNMP 270 dATP 018 0013 ATP 30 28 dGTP 012 0005 GTP 092 048 dCTP 007 0022 CTP 052 021 dTTP 008 0025 UTP 089 048 o The supply of deoxyribonucleotides in the E coli is 1 of that which is needed for replication o In mammalian cells the supply is significantly less o Why keep the levels so low a Inadequate supplies of dNTPs are lethal 0 Too high a concentration is mutagenic Nucleotide Synthesis SALVAGE DE NOVO Deoxyribonucleotides Deoxyribonucleotides H T Bases Ribonucleotides Ribonucleotides Ribose amino acids COZ NH3 De Novo Purine Biosynthesis 533 IJHY 2 Examine and r I S Ipl purlw rihrmlar39lrnhdr hlmywlh m in 1 mlquot Man pull nn nu lunmlcu39 Gum Iminulnvl lilmzx 5PMquot r ATP FHPquhav i I 26 IhnilsluzIJumL39Iaqn39lu lmsplamdl Hl39l Fslff39 AllllklllunLJvuv39 lly quot SCI Pathway is highly u 2quot 33 51y mlm mum 221113 11112 Irrrwlkuuhum z n39 11 CO n S e d m l39mmvlal39nzllurmrln rIbtnlu HL SLKI 11411 IllnllllnL1ulLt Fun Jr fnrvlvy39lul39jvllmmaha nlmlir IF K bacteria to mammals an Inkl hLu v pure 139 lthawamlnJxmlzhxulu riL39ululI L39AJIl I39ACAIH 39IWII Ilml purl 3239 Wh t 7 ml lil Jamsnmuu agnmun iwl l n mum a 3 cu p an S 39 aquot nvlnsmIm Ivuw J m Hmlu39umulmnlu umqu muqu IAII AII MEN unnu I 39ums mm j IIIIH quotII numlnmnzdmuh mllliullzrdll rllmlu IFM quotH Ivudumm i umH I3quot lkll39 ILH39 AHNI39JIrJurTIrJ P ymlum punquot La mlnn Lam cum mvm39llmL 1 Imu lvlmr ma 2 r ibil39 Akaqu Limuu mlk H up unh VIIVIIK391U mm 5 mil rm ATP ILL vI IMN ydmxnzu wuuh M KMI39 nrIhummmumu h a39 lrmxnl quotmy 54 UMP nylm km and cmquot l uvmlrlu diallnwluw tumw nu an IT F39rIn Nmmu mmqu rmkm 33nwwlm Wulmum n w ll 15NN39HHIIIHIJ H IW nwrllnTyl ld39IIlWI39P quotmum u I I Mum Mrruum v m IuUllvlll vxw I First Committed Step in Purine 3 Biosynthesis o First step in de novo purine biosynthesis is catalyzed by 5phophoribosyl pyrophosphate synthase 0 PRPP synthase is feedback regulated inhibitors include IMP GMP AMP end products Pyrimidine Biosynthesis o In contrast to purine biosynthesis pyrimidine biosynthesis starts with the formation of the pyrimidine skeleton and then ribose is attached LLLLLL Jum I rrrrrrrr u Plant Molecular and Cellular Biology Lecture 6 Regulation amp Initiation of DNA Replication Gary Peter 8182008 Learning Objectives 3 List and explain the structure and function of origins of replication in prokaryotes and eukaryotes Explain the molecular mechanisms that regulate DNA replication of the E coli genome Explain the molecular mechanisms that regulate plasmid DNA replication Explain the molecular mechanisms that regulate nuclear genome DNA replication in eukaryotes Explain the molecular mechanisms by which telomeres are replicated 8182008 PMCB Lecture 8 G Peter Origins of DNA Replication 5 0 Specific DNA sequences that are the start site for DNA replication 0 What steps must occur at the origin to initiate DNA replication Recognition of the sequence 2 Melting of the DNA 3 Assembly of the replisome complex 4 Initiation of leading strand synthesis A 8182008 PMCB Lecture 8 G Peter i quotquotmm f i I ll x fi i bt 13939 39 J t i all Plasmids rep 3 HI 0 Extrachromsomal E 1 3 13 orquot DNAgenomes usually g i 3 I I I Il Circular With survrval h H and propagation rig functions including 39 we Replication contrOI Fig 1 Schematic view of the development of survival functions of a plasmid The birth of the plasmid is shown as the appearance of O Partitioning a selfreplicating circle Subsequently gene blocks with specific functions are first recruited by the plasmid and then shuffled by O MUItlmer reSOIUtlon recombination with fitter combinations being constantly selected from the pool Gene names are rep replication cop copy number 0 Post segregation control mrs multimer resolution par partitioning psk post segregational killing tra DNA processing genes for transfer trb Conjugative transfer matingpair formation genes for transfer Molecular Microbiology 2000 373 485491 8182008 PMCB Lecture 8 G Peter 4 Control of Plasmid Replication 9 o Replication is central to the control of a number of important plasmid properties HOST RANGE COPY NUMBER INCOMPATIBILITY and MOBILITY o Some plasmids are able to replicate in a limited number of bacterial species they have a NARROW host range Examples are ColE1 pBR322 pUC18 plasmids which are limited to E coli and some closely related species o Other plasmids are able to replicate in a wide range of bacterial species they have a BROAD host range o The number of copies of a plasmid can vary from 1 the F plasmid to over a hundred pUC18 This number is a property of the plasmid itself and depends on the mechanism by which it regulates its own replication 8182008 PMCB Lecture 8 G Peter 5 Plasmid Copy Control 5535 Systems SEquot o Negative control systems inhibit the initiation of 8182008 DNA synthesis Three general classes each of which depends on the kind of negative control system employed Directly repeated sequences iterons that complex with replication inhibitor proteins Rep Antisense RNAs that hybrize to complementary regions of an essential RNA these are called countertranscribed RNAs ctRNAs Both a ctRNA and protein together PMCB Lecture 8 G Peter Molecular Microbiology 2000 373 492500 Different Mechanisms of Copy 333 Control 9 a 7 ii ian U iiiii iiij gjlmii i Col E1 E coli specific I OriV antisense RNA R1 broad host range OriR antisense RNA repA protein SC101 narrow host range Iterons CoILbP9 Antisense RNA RepZ httpwwwmuncalbiochemlcoursesl4103ltopicslplasmidshtml 8182008 PMCB Lecture 8 G Peter Structure of Origin Regions in gg39r lteronRep Controlled Plasmids mnFi 1 1T l h b b h h 39 MM 0 A 100300 bp region mm termed the control or r I39I39 F ll rep V EV E P inc locus contains the m criminal in p I mm ELquot P K r 7 31 39 J39 o Iterons are an array of Egg If p 20 bp repeats that take FE mme l W3 IIl ITIquotIW patina Wm Emma tom F Deltat shard IF up 12 of the origin Fig 39l ingm mgrm D somc lemmaJim normcmigng 0 Rep proteins bind to these 20 bp repeats 333333 112533 E g cm sequelcam aside bu mom1on ongn and some 15 napemits quotcr Mdma39m n HTch gun rm mu hrmrl 3941 1mm 5 no H In ongn lastrman some 5 MIKE cr m ram gene ll39n quottuners am 1145 EIWMdmtcp39 or H42 Molecular Microbiology 2000 373 467476 8182008 PMCB Lecture 8 3 Peter 8 Autoregulatory Mechanism of Copy Control by IteronsRepA it f E I l Ha 39 W e I D n I I I LOW concentration l LI TILh MP of plasmld and REDA x v j r1701 39 RepA binds to on Origin Drum hmdmg mu Increasmg concentration of plasmid and RepA RepA binds to iterons s s 7 39 9 4 i d J A I f I V V f v v i W gtltllgt IlHmh D i h p PrqIA High concentration of plasmid and RepA handculfing prth repsl ngm OF I 8182008 PMCB Lecture 8 G Peter 1 Saturation of origin Reruns with 2 Replication l 390 3 Replicationinduced rep ll EII39ISGI llplIOI I 4 Handcuf ng chapamne activation or 5 Increase In Rep due to newly synmesrzed Hep l 6 Reversal of handcu ing due to increase in cell volume and In Flap Fly 3 A model shorting the role of Flap prmen 11 he replicatm cycle 01 plasmid PI In his model replicamn xs actmated by 39ncrease n Flap and timed by harncuifngl Hep saturation oi ongn harms notonlyellecls Initiation butalw represses he rep promoEr hat maps wnhri the or ign item aumrepresalonli and 3 Heplrcartm activates Ire pronrow by clean19 the herons of oreexisting active Flap gels marred by daugiter origin which allows handcu ng and promoter repressm and 6 Handwrier is ellm vely reversed by unease in cell volume and 11 mile Hep 011mm may also hep 39n Hep activation as the active Hep gets inactivated tmn concentration in vim W39rckner at at 14291 Requirement hr Hep activatbn could be a mechmlsm to delay hmar m Addrbonalbv Flap saturation oouldbe slow in wewol s erlc hindrance from handcu 39ng Mulrhopacirryary at at 1994 Arti cial hcrease n Flap only slums a modest ncrease n oopynurber apparently because even a mdest Increase rs copy nunber beyond the steadystate value hareases the strength of hancher too much Ior Flag to uni str39ngervt or strumlibs control In this model Flap acts as a posting regulator only at or belowthe steady stale mmemramn oI ierons Molecular Microbiology 2000 373 467476 Control of ColE1 Copy Ct RNA and Rom Protein 0 Replication mediated by the synthesis of a preprimer RNA RNA II and the hybridization of RNA II with DNA template strand at the origin of replication cleavage of RNA II by RNase H provides a 3 OH for DNA polymerase o ctRNA is an antisense RNA complementary to part of RNA II and 0 ROM enhances the binding of ctRNA l to RNA II 0 RNA lRNA ll hybrid alters the 2ndary structure of RNA preprimer inhibiting stable formation of a DNARNA 8Hg hybrid which inhibits replication R Mb II proprimer r I I mm Origin rcm RHAI I Ram l 39 4 Origin K uj lLRNMIWH Ruzrn I I I J No 014 er hybrid J Roplicalicn Inhibllml Fig 3 Copy nLrnber mntrol 39rr CelEl Symhesis of he pnspriner FINA II by FINAF apple1 Circle is essential tor replication In the assetnee v31 irrieractm with the FINA ten the FINA ll forms a stable hybrid 39m39th the template DNA at the orig39rr of replratien Titre hybrid is cleaved by FlNarse H t generate Ite 3quotDH end at Ite Flt134 priner than which repltaltm starts Interact between the nhbitnr RNA l and the EmplEIT lEthal39f regijn 39m the RNA ll brepr39l39ner right is aided by Flam prJ e39n ell39pseL FINA lFINA ll hieraan hintit39s the formation ol the DNA Fl NA ll I39mbrat at the erign region preventing maturation ottne FINA ll into its replissmn priner Comma as 39n the legends to Figs I and 2 PMCB Lecture 8 3 Re er Molecular Microbiology 2000 373 4951500 Plasmid Incompatibilit o Two plasmids in the same incompatiability group cannot coexist in the same cell o Plasmid copy number control is mediated through the action of transacting molecules those from different plasmid types with the same control mechanisms could control each others replication eventually leading tooss httpwwwmuncabiochemcourses4103topicsplasmidshtm R386 Rt Col BK99 Col B K166 R124 R62R64R483 at least subgroups R391 R746 R724 R401 Sa n in m Regulation of DNA Replication in E coli EE fully EEELE ES Fsyswt 7 r methylated hemimethylated origins are origin resistant to initiation i Initiation of chromosomal replication 1 Activation of the replicase quot Formalion of the slldlng clamp WW a draws quot initiation occurs if sufficient origins become fully 7 39 resources are available to complete methylated maklng them at39quot in 4 quot a round of DNA replication again competent for initiation im y Inhibition 7 Figure 5 32 Molecular Biology ofthe Cell 4th Edition Katayama Tsutomu 2001 Molecular Microbiology 41 1 917 o ADPDnaA to ATPDnaA and back again 0 Hemimethylation o hemimethylated oriC is not active for DNA initiation insuring only one round of replication 8182008 PMCB Lecture 8 3 Peter 12 Initiation of Bacterial DNA Replication 39 iiinah Email hain 5 o The E coli chromosome is circular and contains one origin of replication 0 OriC 245 bp 0 3 tandem ATrich 13 bp repeats o 5 DnaA protein binding 9 bp repeats o 11 GATC palindrome sequences that are methylated by DNAadenine methyltransferase Dam o The initiation of leading strand synthesis is the key event 8182008 PMCB Lecture 8 G eter 13 FEMS Microbiology ReVIewsP 2002 355374 E Coli DnaA The Replication 53 lnitiator Protein 0 DnaA is a 52 kDa protein that is present throughout the cell cycle 0 ADP or ATPDnaA is a sequence specific DNA binding protein that binds to 5 13bp recognition sites in oriC o DnaA binds to membranes where acidic phospholipids can release ADP from DnaA in the presence of oriC to exchange it into the ATP bound form 0 DnaA with ATP bound is the active form in replication 0 ATPDnaA bends the DNA 40 then more ATPDnaA proteins are recruited through cooperative binding to low affinity sites located in the AT rich DNA which melt this region through binding DnaA also helps load the DNA helicase dnaB onto each strand which recruits DNA primase and DNA polymerase III binds to the melted DNA to start DNA replication on the leading and lagging strands 8182008 PMCB Lecture 8 G Peter 14 IllIV Junction 347 296 DNA A Structure w mm ma ame AWE B AAEOIKIDS Econ 1 ul hi Yrclkvcs s NAMALA ELINHS I11 47634773 R e39 MJBQer T 21 Erzbergerpgjag 8182008 Mutations in DNA A That Affect its Function V312V383 V3193 V34 61411 1234411415 53255395 T33ZV403 MSAUW 163011101 R3 391407 53761114 7 S 372M A131AIB4 3030156 mauma A104V 57 v143 119 HHSma H197H252 8182008 Table II Lisi nI39E1 1li39 Dnm i 111111111115 Lind i1eir equivalent Auxultras residues 5111111111111 mli mun1111111 allele equi Aquifi l ucnli In In vim defect v leni In rim Lie fact Raf e Ilce CIMSIC mutations Ala1V HZ jPX 111111111401 l4quot 14165 tlmu A 34V P2960 1111111111111 1602 A V 11111115041006 Y2 1H 11111111111111 291311 dun 7 ABLHI 39I Replicuiiun ovei39iniliuiion Cold 111111 Hnm en u 111 119921 Hansen 11 1 31111111 139 al 1987 GilleKcklldelill39i 1 1L IV5 311111111 111 111111111111 11111111 111311 1111111117211 K362 Cold 1111 e DNAbinding lefeci Elnesiiu a 111 1200111 511111112 111 1111e1u11111e1111111 11 111 119951 11111er 5111111 msx 1111111711 391393114 11111 11111 01111111111111 11am Blaming 11 111 120001 SumN1111 n1 d1mx139r11 11111111 iiiKuCaiuielni39iu 11 w 1199311 811111111 11nd Knguni 11997111 042115 mss NA 01111111111113 111111 1 11111 K gnni 1 1911111 Answ A13 Asynclnnnous iepncmion 11111111111111 defect gum 1 199111 1111111111111 1111111 11 111 11911111 11111111111 HI 11751 12 zillelm NM 31111011 and Knguni 11007111 1177 11 4 allele NA Simon and Kngnni 11997111 111341 11131 1 111a NIA 511111111 and Kaguni 119117111 1711 K 21 Noncomplemenling 11101111111111 and 11111 Mimninm a 111 1199 1 mm 11111113 11am 1mm 111 so Noncomplementing 111139 111111111111 11111 quot11 Mimiiimn 11 111 119931 11111111111111 1mm 1199111 11143 1 111m Then 111 51111111111111 K115111111 1101151 131111 r245 3915 iiieie Tlvennolnhile 511111111 11111 Kuguni 119951 113111 1271 mini1111111111111 Detective ATP 1111111111111 Nisllida 111 111 120021 Domain iv 3911 NIA DNA11111111111 defecl Bluesing 11 111 1200111 11411111 NIA DNAbinding 11am Blaming a al 1200111 V403A NIA DNAbinding new menu w 111 1 00111 11411111 NM DNAbinding 1 vi Blacsing 11 111 1200111 0401111 NIA DNAbinding 111nm Elnesing 11 11 1 101 114155 NIA DNAAhinding 11mm 13111611151 11 111 120001 L417 NIA DNAbinding 1m aiming 11 12011111 T436 NA 01111111111111 uem 1511121111 0001 A4401 NM DNAbinding c1 L39ieiing 11 111 120001 K443l NM DNAbinding letecl Elaesing 11 a1 1200111 1147111 TS 111m DNAbinding 11mm 51111011 111111 Knguni 110951 Plienmypic defecis are indicated for 1111111 13911 vim and in 111111 undies 39 S 61 ei39alure wmiliw NA dim I101 available I PM B Lecture 8 G eer Erzberger et al 2002 EMBO J 21 47634773 16 Dnase Footprinting amp Gel Shift for Localizing DNA Binding Sites RNA polymerase ac repressor No protein 5 Sequenceispeci c sequence 5 binding protein 3 il i0 3 5 339 5 Foolprinl C A20 i 539 3 5 3 i Si 0 D E 339 539 339 539 39 1 539 339 539 339 r I Z O gt 339 539 339 539 a 1 r g lt 539 339 539 339 10 3 g rmn 2 m 339 539 339 539 o rmquot z 1 Footprint Fooxpn nling Sequencmg lanes lanes 8182008 PMCB Lecture 8 G Peter 17 Model for DNA A Assembly at Ori C F ig 5 Model fim39 Duxm assembly M1 writquot 39lbpl will is recognized by Juan red green and yellow and architecmral factors surch HF and HLI pulplejk Middle C ummitant with cwrr39C binding the AmiquotL1 d0 maius oligamerize stabilequ U16 uuclenpl39o39teiu mmplex u39nugh inter mmmmel39 contacts ammld the A39lll JJ Lndhlg site Additimml stability may be pmvidecl by dnmain I selfnllgnmerimtinn light blue Bn39lmml Selfasaemhly of DnaA mnlecules eveumally leads a Emma tion of the complete uuclenpl39oteln complex Note that the Una align mer muld conceivably accommodate either a chased ring Ijlya fl or a helical lament right an39angemenl 0f monomers DUE opening may DCC LH spontawenusl Llu nugll lmal strain induceEl by amiemhly39 0f the nuclenpmteln mmplex in the preselme ofquot 2111 8182008 PMCB Lecture 8 G Peter Alternative Models for Bacterial Initiation um nm We origin ncu nl on O O 1 AW mun wwwn w helium 1mm 41 game primaii WIN Inlulnn g man a mm m quotNun L WI 39 t l m m holmuzymn bidirectional re callnn m n inlimwqucafh um replmanon ongm parental DNA helix BINDING OF INITIATOR TEIN T0 REPLICATION ORIGIN DNA helicase bound to helicase inhibitor BINDING 0F DNA HELICASE TO I 1quot ATrich sequence 6 P initiator proteins INITIATOR PROTEIN helicase LOADING OF HELICASE a ONTO DNA STRAND HELICASE OPENS HELIX AND BINDS PRIMASE TO FORM PRIMOSOME RNA PRIMER SYNTHESIS DNA primase ENABLES DNA POLYMERASE TO START FIRST DNA CHAIN a INITIATION OF THREE DNA polymerase NA ADDITIONAL DNA CHAINS begins leading AN FORMATION strand of fork 1 OF REPLICATION FORKS 0quot E P ie tr i Wim Lo39 ELRGQG39d EDIEFQ IE I E quotLL39S I Figure 5731 Molecular Biology of the Cell 4th Edition R primer FEIIII OIIIIicrobioogy Reviews 26 Q n g g 19 Common Framework for Regulating DNA Replication 555 A B C m Kl um ATPwIE 3 U 1 n m 3 quot aroma ll39ln39 ass mmy r oi n un t EFKEE m 539 altInn r quot1 IL 1 IIIL I V ITEHON DNA quot magma 1 AH DNA lpr Ngdlng qr5 li siz39a395huw m M V l39lElf iMv SEENi EFMFI i39l39E b l I u 1 39 g 7 mnnnnlnw mini 1pr A s plasmid Veasi 20 FEMS Microbiology Reviews 26 2003 533554 0 Yeast Origin of Replications A 55 00 Model On for Eukaryotes 3 o Origins spaced on average 30000bp o Permits replication of a chromosome in 8 min 0 Origin contains the binding site for the multisubunit origin of replication complex and binding sites for other proteins CHROMOSOME Iquot origins of replication telomere centromere telomere 100 200 300 I nucleotide pairs x1005 I Figure 5 37 Molecular Biology of the Cell 4th Edition IB1IB 2 site paws Figure 5 38 Molecular Biology of the Cell 4th Edition 8182008 PMCB Lecture 8 3 Peter 21 Conserved Structures of DnaA and CchQrm 353 Cch a A M Orcf Erzberger et al 2002 EMBO J 21 47634773 8182008 PMCB Lecture 8 G Peter 22 Mechanisms of Initiation of 0 I I O Eukaryotic DNA Replication O o A similar sequence of events is required for initiation of DNA u allquot replication in eukaryotes a WINquot 139 0 Due to the larger size of the a genome in eukaryotes multiple H origins exist o The initiation proteins are M normally bound to the origin across the G phases of the cell cycle 0 Posttranslational control phosphorylation initiates DNA replication in the S phase coordinating the firing of multiple origins on each chromosome 8182008 PMCB Lecture 8 3 Peter m min mile run assembly 3 0 5 Dru 39 i w quot an mm ATPH i mgrling Preinitiation Complex in Yeast Egg 0 Origin Recognition Complex 0 A six subunit complex in yeast that binds to the Ori sequences miman magiquot DEC 0 Other Prereplicative Complex g Proteins quot9quot o Cdc6 binds to ORC and recruits Edm1393 439quot can Mcm proteins to form the preRC o Cdt1 cdc1O dependent g l o MCM proteins are helicases 0 Licensed Origin contains afully 1 v assembled prereplication f complex waiting for activitation by MOM S Cdk together with cdc7 kinase triggers DNA replication Licensed origin 8182008 PMCB LectureB G Fge nes to Cells 7 coo Control of DNA Synthesrs by Eggs SCDKs 533 ORC origin recognition complex DNA 0 Initiation of DNA replication Ogbfingsi e o Cdc6 binds to ORC and CM 3ch recruits Mcm proteins to form G1 f the preRC I 653 o S Cdk together With cdc7 Mm prerreplicative kinase triggers DNA 39 Complex ipreRCi replication by recruiting cdc45 awkwgggns Odes g gggg w gi n ch to the MCM complex and SP HASE p inducing unwinding The DNA PngHORYLA w OF RC polymerases 0i and 5 bind to start replication 8182008 PMCB Lecture 8 3 Peter 25 Coordination of Activation of Replication Origins in Euchroma First Before Heterochromatin 0 Pulse labeling experiments show that different regions of the chromosomes are replicated at different times 7 Dark regions have replicated in the window indicated at the bottom 0 Heterochromatin late in S phase X chromosomes inactivated one is replicated later than the active one early 8 middle 8 late S 0 Increased histone acetylation 02 hours 35 hours 68 hours directly causes the earlier firing of replication origins in yeast Figure 5 35 Molecular Biology of the Cell Summary 0 Common steps in initiation of replication are 8182008 Origin of replication short stretch of DNA sequence Recognition of the ori by DNA binding proteins Recruitment of other enzymes involved in DNA synthesis Helicase activity is the most important first step PMCB Lecture 8 G Peter 27 Plant Molecular and Cellular Biology FOR5530 F Altpeter Agronomy J Davis Forest Resources A Hanson Horticultural Sciences G Peter Forest Resources Plant Molecular and Cellular Biology Lecture 1 Course Overview amp Intro to Recombinant DNA Methodology Gary Peter l 99 quot Ecoli m m human 8182008 Learning Objectives 53 1 Course Objectives 2 Module 1 learning objectives grading and expectations 3 Use the fundamental amp powerful conceptsframework of molecular amp cellular biology 4 Apply biological reasoning amp evidence 5 Explain methods and approaches that are usedneeded to elucidate molecular mechanisms 8182008 PMCB Lecturel G Peter Course Objectives 8182008 Understand current knowledge of plant genomics and fundamental molecular mechanisms that mediate plant growth development function and adaptation Understand experimental methods and strategies used to elucidate molecular mechanisms Promote students ability to interpret and design experiments to elucidate molecular and cellular mechanisms controlling plant growth development function and adaptation Enable students ability to read and analyze primary literature in molecular biology and genomics PMCB Lecturel G Peter 4 Course Introduction 53 0 Syllabus c Expectations o Universal Intellectual Standards 0 Clear o Accurate o Precise 0 Relevant 0 Depth 0 Breadth 8182008 PMCB Lecture 1 3 Peter 5 Learning Objectives for Module 1 53 0 List and explain the concepts molecular mechanisms and proteins and their functions that mediate DNA replication and repair 0 List and explain the mechanisms that regulate DNA replication in prokaryotes and eukaryotes 0 Apply the principles of recombinant DNA strategies and methods to investigate the function of genes involved with plant growth development and adaptation 8182008 PMCB Lecturei G Peter Grading amp Expectations for Module 1 53 o Expectations 0 Actively engaged in learning material 0 Check for extra information posted on course website 0 Use of correct biological terminology reasoning and sufficient level of detail 0 Grading o 1 Exam worth 50 points 0 4 Homework assignments a total of 50 points 0 Assignments will be posted on the course website Problem Problem Problem Problem Set 1 Set 2 Set 3 Set 4 Assigned 825 91 98 915 Week 5 Exam Due Date 91 98 915 922 l 8182008 PMCB Lecturei G Peter Time seconds Scales of Analysis E E Primary Biologists39 Focus O O 2quot Primary Industry Focus 039 1X 10399 1x 105 01 10 100 10000 Space meters 8182008 PMCB Lecture 139 G Peter Fundamental amp Powerful Concepts 3quot of Molecular and Cellular Biology Replication DNAgtRNAgtProtein Regulation StructureFunction 8182008 DNA synthesis lt ireplicationl RNA synthesis transcription protein synthesis translation amino acids Figure 1 4 Molecular Biology of the Cell 4th Edition PMCB Lecture1 3 Peter 9 What Do We Need To Elucidate Molecular Mechanisms 0 Understand the structure function interaction regulation and organization of molecules that mediate a process 0 Way to identify individual and groups of genes RNAs and proteins critical to a process o Way to manipulate individual and groups of genes RNAs and proteins to affect the process to dissect their rolesfunctions in organisms 8182008 PMCB Lecture 1 G Peter 10 What Constitutes Evidence for a Particular Biological Mechanism 0 Proposition 0 Genetic and biochemical data are the only kinds of biological evidence 0 Genetic evidence permits identification of genes involved in particular processes and provides in vivo functional evidence in the context of the organism 0 Biochemical evidence permits identification of genes based on in vitro function and provides detailed understanding a protein s reaction mechanisms and mode of action 8182008 PMCB Lecture 1 G Peter 11 Example of Genetic Evidence for the Function of a Specific Gene 0 An organism with an altered phenotype is iden ed o The mutation which causes the altered phenotype is heritable and segregates in crosses between mutant and normal wild type nonmutants 8182008 PMCB Lecture 1 3 Peter C O O O O C C I O C O O C O C C O O Firs Eeneralion 0 While tww 1 quot If r r Purple x R cm 39anl39 quot 39 Second Generation N x El Purpxiale I a 39 Ix 1 MW WM r Pmple le Well imi Example of Biochemical Evidence 3m for the Function of a Specific Protein 5 o The association of specific proteins during purification to homogeneity with a measurable activity A h a E 8182008 PMCB Lecture 1 3 Peter Combined Genetic and Biochemical Evidence Genes amp Proteins Responsibl for DNA Replication in E coIi quot 0 Forward amp Reverse Genetic Screening 0 Temperature sensitive mutants impaired in DNA replication 0 Quick stop 0 Slow stop 0 Biochemical Reactions 0 In vitro reactions competent for DNA replication o Complementation 0 Purification o Subunit structures 8182008 PMCB Lecture 1 3 Peter 14 Biochemical Approaches 53 0 Specific assay for activity of interest Develop with crude extracts a Test for stability of activity 9 Conduct single or multistage purification of proteins or protein complexes o Isolate and characterize protein structure and function PROCEDURE TOTALVOLU ME TOTAL PROTEIN TOTAL ACTIVITY SPECIFIC ACTIVITY InL mg units LInitsfmg LCTIIdc extract 2000 15000 150000 10 2 I immonium sulfate precipitation 320 4000 140000 35 3 Ionexchange chromatography 100 550 125000 227 all Cell iilralion L hI39omatogrrmhy 05 120 105000 875 5 Affinity cinematography 8 3 T5 000 15000 8182008 PMCB Lecturef G Peter 15 Genetic Approaches 55 0 Forward genetics Looking for mutations in natural or mutagenized populations that cause changes in phenotype 0 Selection 0 Screening 0 Reverse genetics Creating mutations in selected genes to determine their function in a process eg Shuman amp Silhavy Nature Reviews Genetics 2003 4 419432 8182008 PMCB Lecturel G Peter 16 Development of Molecular Biology donor DNA vector 0 vector and donor DNA l 322333 33 112333 tquot l o Recombinant DNA n AA Td methods evolved AATT Ann mixing AquotTT m n In DNA ligase added seals alternangs I I AATT An bacterial genetics G g MTT T A ATT and biochemical Bi in iies chromosone DNA introduced into bacterial cells StUd39eS Of the e n y D recombinant DNA molecules replicate and cells divide u 095 zoo2 Encyclopaedia Britannica Inc 8182008 PMCB Lecture 1 3 Peter 17 Manipulating Molecules Creating Novel Sequences 53 0 Isolation of unique sequences 0 Synthesis o Chemical synthesis 0 Cloning o Plasmids Phages Polymerase chain reaction 0 Transformation Transduction Conjugation 0 Restriction enzymes Ligation Recombinases o Amplification of unique sequences 0 Plasmids Phages Polymerase chain reaction 0 Selection 0 DNA sequencing 8182008 PMCB Lecture 1 G Peter 18 Cloning amp Amplification In Vivo 0 DNA replication 0 Plasmids 0 Low copy 0 High copy 0 Origin of replication 0 Phages 0 Double stranded 0 Single stranded 8182008 0 recombinant DNA molecules INTRODUCTION or PLASMle INTO BACTERIA Figure 8 33 part 2 of 2 Molecular Biology ofthe Cell 4th Edition double stranded recombinant plasmid DNA introduced into bacterial cell bacterial cell cell culture produces hundreds of millions of new bacteria many copies of purified plasmid isolated from lysed bacterial cells Figure 8 31 Molecular Biology of the Cell 4th Edition PMCB Lecture l G Peter 19 Putting Novel Genes into Cells Transformation tquot Transduction m B l IPMMM v lltr mlkl 39 I air1r Conjugation Selection medlibmedutaheduFigures Lecture3ConjgtnJPG 8182008 PMCB Lecture 1 3 Peter 20 Amplification In Vitro 39 o In vitro reactions o Purified DNA polymerases Polymerase chain reaction Polymerase hain Reaction PER 1 Eli r39 1 1quot u i quotF r 4 quotL i r39 i Cutting Apart amp Putting Back Together a Restriction enzymes 0 Methylases o Ligases 0 DNA double strand 0 RNA single strand Eco RI enzyme DNA complex RosenbergJM 1991 Curr Opin Struct Biol 1 104110 Review of EcoRI Studies 8182008 PMCB Lecture 1 G Peter 22 Example Cloning a Gene 0 Need pure plasmid with selectable marker 0 Restriction enzyme 0 Ligase 0 Transformation method 0 Methods to analyze inserted DNA 8182008 PMCB LectL Foreign DNA reg39on ofmterest Plasma 39 ECDM EcoRl 1E RI gene for antibiotic resistance EcnRIl lEcoRI Sticky ends39r Hybridization l DNA ligase Recombinant DNA DNA in 2 Iliiesl Baunma B gotcha Bacteria plated on medium k v c omosome u antibiotic 4 quot3 Only bacteria containing I J recombinant DNA grow I Culture 1 DNA uni cation 0amp0 Cloning into a plasmid History of Molecular Biology 55 0 History of Genetics Timelinehtm o HttpwwwaocessexcellenceorqAEAEPCWWC 1994qeneticstlnhtm o MolecularBioloqist com A Concise History of Molecular Bioloqv amp Geneticshtm o HttpmoecuIarbioloqistcom 8182008 PMCB Lecture 139 G Peter 24 Summary 53 o Fundamental amp powerful concepts of molecular and cellular biology o Replication o DNAgtRNAgtProtein Central dogma Regulation o StructureFunction o Genetic and biochemical evidence 0 Recombinant DNA methods exploit natural processes for the manipulation of genesproteins 8182008 PMCB Lecturei G Peter 25 Important Resources 3 1 Databases available on web 1 NCBI 2 TAIR 3 JGI 2 MethodsProtocol Manuals My Favorite Oldies 1 Experiments in Molecular Genetics by JH Miller CSHL 1972 2 Guide to Molecular Cloning Techniques ed SL Berger AR Kimmel Methods in enzymology v 152 1987 8182008 PMCB Lecturel G Peter 26 Plant Molecular and Cellular Biology Lecture 3 DNA Polymerase Structure and Function Gary Peter 6299 239 82 8239 xx krxxg 82 8299 A 8239 82 AKlt29 8239 REPLICATION EPLICATlON REPLICATION Z2 1A Figure i5 Molecular Biology of the Cell 4th Edition Learning Objectives 5539 newly synthesized leading strand 1 List and explain the mechanisms that insure highfidelity DNA replication 5 2 Describe and explain the 39 t structures and functions of gm fesized Begin a the enzymes responsrble a in helix 99 9 for DNA replication H strand B c Figure 5 22 part 2 of 2 Molecular Biology of the Cell 4th Edition DNA Polymerases Function amp Activities 0 FUNCTION 0 DNA replication amp repair 0 ACTIVITIES o Polymerize dNTPs along a DNA or RNA template 5 to 3 of the growing strand Processively o Exonuclease 5 gt 3 o Exonuclease 3 gt 5 i E COLE DNAPOI I 5 CAP 2nd Strand CDNA Synthesis AAAAAA 3 TTTTTT 5 mm 3 TTTTTT 5 DNA Polymerases Fall into Gene Families 9 0 Gene family identification relies on DNA sequence information and functional characterization of activities in vivo and in vitro o Different family members have diverse functions during replication recombination amp repair Table 1 Representati39vre members of families of DNA polymerases Famin Prokaryotic Eultaryotic Archaea Viral A Pol Pol T3T5T7 pol E Pol II Pol 11619 Pol BI Bil AdenovirLis HS 39 F369 T4TI3 pol I P01 Illita ji D Pctl D X Pol flu Tdt RT Telon erase Reverse transcriptases Linquot Linu DinB Pol I u u39 Pol n l h nature structural biolcgy o volume 3 number a v august 2001 Phenotypes of E coli with 5 mutations in DNA Polymerase Mutations identified in the DNA polymerase I gene show that it is not required for DNA replication rather it is involved more in DNA repair DNA polymerase III is the major enzyme involved in DNA replication ABLE 410 Phenotypic defects of polA mutants Sensitivity to thymine starvationquot Sensitivity to ultraviolet irradiationc Sensitivity to xray irradiation Sensitivity to methylmethane sulfunater Extensive degradation of DNA in repairing ultraviolet damage Increased frequency of deletions Increased frequency of recombination Low viability 5 percent on nutritional shift up ColEl factor not replicatedf ColEZ factor unstable Mini circular plasmid of E coli 15 not replicated Poor joining 10 percent of 108 fragments Singlestrand gaps in X174 RF l Poor growth of phage A defective in exonuclease B or 7 proteinquot De ciency in phage T4 replication and recombination Gross I D 1972 Curr Top 57 39 bNakayama 11 and Hanawalt P C personal communication Grass 1 and Gross M 1969 Not 224 1165 Kate T and Kondo S 1970 Bact 104 871 Tow n C D Smith K C and Kaplan H S 1971 Science 172 851 Cooper P K and Hamwalt P C 1972 PNAS 69 1156 39Coukell M 13 and Yanofsky C 1970 Not 226 333 Konrad E 8 and Lehman I R personal communication Rosenkranz H S Carr H S and Morgan C 1971 BBRC 44 546 Kingsbury D T and Helinsld D R 1970 HERO 41 1538 Goebel W 1972 NNB 237 67 Gnebel W and Schrempf H 1972 HERO 49 591 Okazaki R Arisawa M and Sugino A 1971 PNAS 68 2954 Kuempel P l and Veornet t G E 1970 BBRC 41 B73 Schekman R W Iwaya M Bronuitrup K and Denhardt D T 1971 IMB 57 177 quotZissler Signer EL and Schaefer F 1971 in The Bacteriophage Lambda A D Hershey ed CSHL p 469 39Mosig G Bowden D W and Bock S 1972 NNB 240 12 Kornberg DNA Replication 1980 E Coli DNA Polymerase Activities and Features TABLE 54 Properties of polymerases I II and III of E coil pul I l nmJum Polymerization 59 339 47 EXLIrIuUIEHxM 339quot 539 l xonunlnasc G vrfl i VIthusphornl5is and PP exchange l TPleaHLpI39Im r uplex Primed single slmntlr Ninked duplex poly IJATJ gt Duplex wilh gaps ur prvlrutling singlcslrnnrl 5 Ends mn nuclnmidcs l mu nucleulides 39i nlymer synthesis de novo Activity 20 mM bu a 90 mM 80 lylfum 1 Mil phrnnnl nf upnmal mu 1 1m l n mM Km ur uiplmphmn luw lnhibirinn by 2 dcoxyanalugs Inhibition by arnliiunsyl 111 7 lnhihilinn by sulfhydryl 5H bluuking agnuls Inhiblllun My lml l millsm39um l in Size kdal 109 Al nlly In phnsplwccllulose mu arily ul39 plumplmln rnquimd for clutiun 0 15 Mnlemlrsccll estimated 400 l39nrnover number estimaled 1 Slruuluml 3am palA Conditional lethal mulanl yes an n 5w 39l39a l ml 11 005 pain no 4 y and r represenl he pmam ml nbsmlcc Icupcmivcly of m pmpnrly 1mm msnnmmn mm 1ml 1 diminmnn bulwunn pl 11 imd pnl m Kornberg DNA Replication 1980 pl lll 120 010 10720 15 pnlu yex A prinwll Minglv sir1nd is lung singln ram wuh n thr length ur cumplomemary eranrl nculcd Ir ll 1 lmlnn mymanmi n Zl7 nuumolccule u cnzymo Mamu m ml L n him is war nut 1 DNA Polyermase Small Large fragment fragment Klenow fragment Structure of DNA Polymerases EE incoming deoxyribonucleoside triphosphate quotthumbquot template strand gap in helix quotfingersquot primer strand B V quotpalmquot Figure 5 4 part 2 of 2 Molecular Biology of the Cell 4th Edition nature stru mural biology r volume a number a i august 2001 Reverse Transcriptase Domain Structure 5 RNA template strartd quotfingersquot polymerase active site synthesizes DNA strand 1 i quotthumbquot direction of RNAse H enzyme If movement Ifquot RNAse H active site x quot TWENA degrades DNA strand 539 5 ran A iBi Figure 5 74 Moiecuiar Biology of the Celt 4th Edition 8182008 PMCB Lecture 5 G Peter Conserved Structure of DNA Polymerases EE c 7 3 3 mm P x lm ETP 2ETP1 ETP dm Ja TPn1 Family A Family B Thumb v Fingers 3w Pol n T7 Pal r l vi 3135 Pnlymennn I 1 complex 313 Molecular Cell Vol 8 417426 August 2001 Fingers rm poll R569 pm


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