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Lecture 7

by: AnnaClippinger

Lecture 7 BIOL-L211 2521

GPA 3.3
Molecular Biology
Megan Dunn

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About this Document

Lecture 7 notes along with extra notes covering the trombone model of replication
Molecular Biology
Megan Dunn
Class Notes
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This 16 page Class Notes was uploaded by AnnaClippinger on Monday September 28, 2015. The Class Notes belongs to BIOL-L211 2521 at Indiana University taught by Megan Dunn in Summer 2015. Since its upload, it has received 38 views. For similar materials see Molecular Biology in Biology at Indiana University.


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Date Created: 09/28/15
El Herder Lecture 7 Sep 15 3015 Last Time Replication Today Mailing Msrqnmem quotuch q pg mu v Z lz 31oz wtato lmrm eh l m MRIla 1695 we Replication Nucleic Acid Basic Methods Next Time Problem set 1 due beginning of class Thurgda 3 cm Willl be ReadingAssignment wand 0quot Hamidl maltrlul Molecular Biology in the News Hello mothers hello fathers 39 Chapter 939 pp 288292 302307 N ucleic Acid Molecular Biology Techniques Replication Initiation tho have a mph wl 6mm 3 rm 0 e armored m r 1 Gquothx W l or the 7 eowrlbugg for l O Wr39 quot r I ewsrmndq mg wand m DNA synthesis overview an Normals renames 1 inan quot 7 j39 L I iuLJrrl I 3939 itquot VI a I I In F rm 6 o or39l39 llldlvldual dMTPS addEd 130 Ei Wing DNA strand based on template strand own to the 39 1 r E 15 1 quotiiili iii ilVV39C39IJIiI I 121i quot quot W v 539rer band Fern term 0i EN PWPh i l t 0an alpha lch n This annd t rill DNA synthesis overview Template Strand determines which dNTP is added Team new To be commmtmea WI 3 OH of primer attacks Lit phosphate 39 of incoming dNTP Ali 0 mi led pm mogpn um tern plate a 539 HO39 39 39w thmetes rammed D Mohameder Bond form DNA polymerase is processive quot 39quot quot5 quotIii ilili miii mf i UH339 DNA paiymeraae binds slow putative nonproeesslva pmmsagw DNA DNA pdlymerasa pawn133353 r E 39 wwnm DNR oiimermeblnds iii a How 1 i prom P i i 5L RN R in mer ma 1L me when Hui Iii 339 min Bi ms dam n DNA polymerase uses on exonucleose to proofread Errors do occur Proofreadin Mispairing aiters positioning of the 3quot OH DNAsynthesis slow i gt w wading tne W rang Basc Changes tnE WWW in its um dawn one mininon removes imp 39 roperlv basepaired le TiPs 0 DNA DUN new a 51M or no DNA synthesis We b removal of infiiernaidieeri anaemiaBis Primerztemplate junction destabilized Moves to exonuclease active site Em nucleate removes own The mun mew error quot Em Wi HueF inn Ii aligned 39 in I Livf a if Straitmsiiijnnnerename 5 W n re ne r E39S39quS39 f f f 39 H as in anon it while t s SH ii I nine Primar i B QS Q 4 nus T0 imM39Qdm rEH CGi39Chl39t w v 393 leOr Bdnwn thE synthesizing leading and logging strands F mm Leading and lagging strands s g the repliea on for DNA DON meme M Emma9 to quot S39ilii riRSquoti 2Q lquot nemtmndi 9397 m 39 3 strands are Inn qmi gi3 2 h in editiamni quot nln39 o e r inureme M m Les ictrandr inn unannanana hiiii 39 tm MW MS goes mine W inn in me since iii Eur on u the we in I i un m at I nq R ED Iiquot39ah39un ink M ditmnnnuwsFyiqgmen WM 1 nus w Mad in thesame direction that the A y I gt 7 i replication fork moves K H U quot39qumtntS flirty Made in long continuous stretches W n11hgrmlr i b 0ne primer quot 2 2 m r a owing g wf strands H Tmulgmp lawful 2 4 U199 inq thaumnmnnls h39 Strum B a m in nu QP39EH up a the replicatmn Purn The E col hieoenzyme and replieome S E Du WGH H To g D l quot w it linkiimmml zi w t WM NH WNW 13M 9 Lla39rrlp Inuldar39w DNA polymerases oriented in opposite directions 1 replicates leading strand 2 replicate lagging Ha rmf n it WE Wm wens in repair acid nth EFF merit a MW j n m DNA mirer l in DN A5 leadingstrand DNA polymerase together at the replicatan fork h umq meruSt Hume Simila r in eu karyotes but less DNA p wmemse Nd39erStOOd Okazakl fragment H wmrtnrul rMultipwime V l i The trombone model of DNA raplica owmch 0m mu 0 B 5 39 Helicase unwinds DNA at the replication fork PUl El a loader 1 r t lquot l t 3quot an amt 5i iiinettle the pm 939quot g W1 WM and slidingh 1 v 1 m t Clamp 39 V a quot 39 i 4th tithe 1 DNA will qu3939859 lagging Strand CW a MS Mm 2quot D 39539 W maze rmtagqm tram pith in l Illlmlgw mm L an39E Strand SSBs coat the ssDNA C39lUClll I DNApgolymerese PilllQlllS regions warring o a Dicaz k fmgmtm I r R d 39 IV V th qu Hm 39 l Okazaklfragme will d liltWt l 39 e Inewi s In eslze nun TD r e Um un V Equot DNA in DNA pol Eeplitgatirt Itadlmmtrusnde rhit 39 Greeri RNA primer Q39 i39 Jm g Figure 9233 i 39n m 39939 3 directi on talamp l 2 lil V leadingstrand V quot DNA pplymerase sliding clamp 39 5539 The trombone modeI of DNA replica ongw Heme reiis s nmwiemwh NEW Juncii 3 1 tiquot 3939 5 synthesizes a new RNA 3 I i i n quot 7 39 IV39 1 u39 I rlmer on the la y in r 7 p legging sirend Pol quot I ra centimues Okezaksl fragment synthesis 5 n n I i r r i iquot I 39ir39iquot assembles a 39siidiing ci amp siiding elempis leaded g i i 39 mm rlhe newly primed quot onto the riewhgr primed lagging strand iagging strand 3 DN MON MQMSQS N Jth mat up io m mm u 3 pri mase u iij efgedw i an tmm A Airs li il i IJ L in me wow um Viaeo is green are pow memes 53 v BUM i 539 inch qu y w m Dark purple ISSiiqu a ale pr liner is NUTg igm s The seeundvquotlag39gingastrem quot 39 a DNApe Iymerese iw bindsthesiiding ciemp at the primeriemiplele iunciien and begins in synihesize anew a quot Okazaki fragment l i V 1 V 39 I g a mquot i 1 l I I I q 39Unm quotmt i ihe new 04 f mg mam B l an Ukezakii regmenl The quot rstquot laggingstrand Figure 9 23d e Finishing repiicon39on V for mom imam i i Tm ENHRDHWIW R pnmho m r 7 7 removed 3 39 na in ow a yDIn Ir n L 1 5 5 one remover momma in rm rm DNA Enzyme warH Wow Wow isr dq H39s an Rm primer ommrae is El DNH prrm c Last nucleotide removed by 51 i w i w H l Em WOMEN remover nutieorrqer from r i V 13 i ii 1 M E 9 7 r Prin glate Ital D W I d b if a have or 393 m mom 1 p 339 WU F 39 fr marl Orr 0130 PhDSPm diQST Qr39 BU i m 11 The end rep coiion problem ofiineorchrqmosomes we a Mira m drama ii are rim ht iriog i ends of linear chromosomes Leading strand 6quot Lagging strand RNase H removes primers repair Dkazaki fragments 39 39 glut rquot n I rr ram 1 DNH PM W1 a r 7 reoiiaatad DNA Sh T i l if hrom or name needs 0 primer J 77 rep cote agan C h rum mime I ij probirm is repaired in new r Iaqqmq S rmrrdr 5 5 5i39I 3quot chromosome is shorter The problem is soived by Fig ea 5 r w replicates the ends of linear chromosomes om osed of protein and RNA n l8 tcm lure a UEBCAa cf S39EIWMquot 1 DNA aiynlhaaia i own RNA Repeats process r g allEDGAAUh aaf39quot tIaa itIAEi twelem39 I DMAay39n iasla repeat Replication enzymes and disease can him many roles in diseasesquot Most have a mutation in WEN a gene encoding r tame Telomerase binds to l w H J w RNamumw bi V iii RNA com momsa nals the amt server Q Q Wxalas bases that are complementary to aw a can run into PrplalEW when mu 510p mg I 139 Decreased telomerase activity has been implicated in the aging process Incience in r 7 I Inheritance Pattern Autosomal Recessive an u 39 39 Symptoms Early graying of hair Loss or w RN also i implicated in Mt l m d telomtmnorrenim uginug str nd 7 Ta tcompr w AW id m is smiling wur N we call on mm m g l V w i mm pillnrt nim 05qu LT39LT Telnmtme f I Slntn39esIzes mu We 339 direction f remarureamq f loaoog USu lN pilth IJUD QYN wrinkling and sagging of skin Decrease in muscle mass Premature loss of stature Cataracts Shorter life span death by 50 a Q a mum on wt i n Tm 394 a mate 14 In vitro process modeled after cellular DNA replication Method for ampllfying small quantities of DNA a Vernon or DNA R pllt mh vs usualw l n PCR we mleremd Ingus39l a gene lomusr a porn on or a lane 1 separate the jch DNA strands none the Ugh new n9 N 0 repquot am on 0 r M f Dr M m lanneel primers a primers to the DNAstrand 39 mp down to 0 WOW W W 5 w 39 he graham of DNRE pnmm use own Prlmlrl en RNR primers an atom Wl ll mln l quot ll39 39 L J a 39 WILILN WSW DNA polymerase extends the pr imersequence Tmall 51mm Ll mun In cunnnuum mem elangate with DNA Balmerase 15 commeli an 0139 all quot38 pS The amount of DNA doubles after each cycle Final amount of DNA 2quot where X a of cycles all llll llll all Oh I all 1T If N mrr M i m mammal DNll one 3 PCR 01 how much DNA NOUlcN have 3916 lheal denature talmtmtmmrinimumnrirmmrmim LS it protein l H III Imlllili it 39 f i want anneal primers m w iTrmmm than um we r We Won quotmu illi llll39llll elongate with DNA palymemse want to 01mm heat and lama Extemi onst MP Nnm pow marate it MlliLHllllllllel lll l t F V anneal primers gt and elongate 39 V quot 39mmnmm squot i 4 w l H mw o Ute s mm DN it put an molt T13 Wlttancl hr 7 7 39 r m 7 m a I it to JUPPH39TD NS 0i d V S lt Emmiquot 0m Witt l 10m l111 are tatPa mm CDHS I emit on mm diam tm dNTPt MOST 10H V Q Flg 712 17 Agarose gel electrophoresis 0 p H a 3k POWEF SOU FEE Method to analyze DNA fragments from PCR or restriction digests Chamber lled with buffer Aga rose gel 13 did mm on New Wl LQC NWT The sliding Clamp increases DNA pelymerase processlvity leth of Multiple subunits Binds DNA polymerase lid NI l nfmm to the DNll l F l quot 7 39 I E39La F F up 3 1 z 39 39 b l direction of tepileallurl sliding clamp 39l template strand 7 13L I l II A l V ll 9 l QUE17W l Iquot V Keeps DNA polymerase from diffusing away from DNA illlil39 51 39 u 7quot uji739fz li 39li l elf7ft q 77 7 template strand m qcld thousands of bases 6 u 1 m 19 The sliding clump increases DNA polymerase processlvity I a qu will mag me 5 lef DUN mam jummn 45 mm ISthlr Dlrclmq DN ll 0 it Ti39ltS lt DQCKQ a sllwing damp template ID NA wtymemse 39L 171v 39nrimn39lmmilm in l m 1 v 39 5 V I I I l Him12 IliaiiHEIHI HTHFHHHEH w Sliding clamp prevents DNA polymeraee from 5 completely falling off a ma DON mU SQ twmas can nuu DNA 39 Mn M 91315 t plumJar to PTW M quot 39 l nmn39a39umm mulls Fig 940 20 Primer synthesis in eukaryotes DNA Pol w primase RNA primer 1 I synthesis by pr imaas l w v l g lllllll a f lllll 39wsmrl DNR D Syithesizes primer quot 10 hp then falls off Does NOT mmatt mlx39luletl m W WPFORSSWW 21 The sliding clamp loader loads sliding clamps Sli djingclamplnacler I N mmml will i H 39 Will 39COQHIZE39 summating PhNIi lCle apemt ATPbound clamp loader binds and opens sliding Clamp H w m allm cl DH MQH remp39l 39 NH Jun CHOW DIG CBS35 l1 ding lamp 3 pr mar 1mm p lure lunar on 393 ATP hydrolysis releases clamla loader from DNA and 7 r l 7 m 39 quot390 sliding clamp H39Ud r bl n k cps Hquot from Flaming may Sliding clamp spontaneously closes around DNA 22 Oi DNR mPliCOhOii Synthesizing leading and logging strands Leading and lagging strands synthesized simultaneousiy atthe replication fork DNA POW MGR SE REfiD i39EmPia PE 3quot 9quot QNIiangrLzeg new 103 direction of leading strand polymerase movement leading strand overall direction of DNA rapiioation atquotl quotIoaft 192quot quotLi12quotquot H iii6T6 5 539quot L L i i 1 1quot L I 1 r a 5 v a i quot37 ll 4 r 373439 5 J 35aquot if 39 FratJ a quot 339 fin39L i 4 Lju 1 t39 3539 pquot isy un hm tagging sireLo aquot 4 direction of agging strand polymerase movement I e 4 ii reipiicated Honanimated DNA Leading strand Lagging strand Made in thesame direction that the M dE in horh GUS WWW qu39mm ff replication fork moves m kazam agmh Made in long continuous stretches Mum pmmm One primer t me 1 huw lw Cam Rtpll dclr ltaal m E lagging The E coli holoenzyme and replisome 5mm mm ulmmguw ma pummemte l f N M ul pmr m Compl x i F Holoenzyme 0 Calm CW rpm Sl mm link 4 rue1mm Cal l m 4 M V 71 0me pow mrme 1 3ndan Eldan low lav a DNA polymerases oriented in opposite directions 1 replicates leading strand 2 replicate lagging strand sliding clamp x i loader I39y sliding clamp FE922 a 5 a w quf x l sA ithchmp 7i w All of the proteins acting loader rprotein together at the replication fork 39 quot 39 form the replisome g Glam r DN pON MENSi them are pearl OE We Rt MfSOM Q unengeged DNA l U El Ether THESE Prom n form a 9mm P ll merase 39 tuned forDNHSWWESIS 3 u w lndwd alwtm WClelQJS RelFunny J PQY all g QClFl C Bing r brow n la ginnglstra nd quot Sirnila r in eukaryotes but less a a My VD A p oquot l m e rasquote understood 000ml mail ed m we v A A Dkazakiiregment ll l Ttru tliom berm em leeding strand DNA Polymerase ALWMS Synmaize m quotif ChCiP i EX Ci The 31 direc on w w a The trombone model of DNA fplica on 1903 Ef gllgggg glqwfcatmgm 0W quotOWEN fine i Helicase unw inds DNA at the replication fork w w ieedingaetrend M w poi merese i y g sliding clamp V I w F 0 POI EHZVW loader I pmtein WDNAPDIE Curb gymm Size S leading a l D 993 39 We 939 I 3 539 w i Rreni m quotWit thgr39 2 DNH Fbi 39 Tunas a z mg gwhmefm WE m3 eiamp A v unengaged DNA poiymeraee L39 e lt r as bound Prima e WW Ewing strand SM 3 mifnnm a 5535 coat the ssDNA 5N dawn 0i g DNAgoiymei ae E mnmm aw g mar 0h i c mgmmf AMEN mm k er ems iquot regions Sigimd quotRM We 39mWaqgme Swami p fn n httd RNH itmi w m Okazaki fragments iv Red newly synthesized f i F Si w cmalw WWF may on 1 omen iii Ema mm aii m DNA a 05 mm goes Lindy w w an a 51 mealc mm w Tei agi gimles take539 6306 W m dWiiEWi i id N Wii imu ud ram Green RNA primer Figure 91233 The trombone model of DNA replication 1 wan to explain wow wow can now pmg mitmgtwnsrcam w b CD rm EC ttd TD CitI M wortmc F51 mus Tone LuSw a h e H 131 I s Commian quot 5 quotmowm EN Pi Wim isw associates synthesizes a new RNA primer on the lagging i P t C iagglng strand Poi 5 ran giRNi primer 39Thnz k primese 39 1 1 RNA primeris synthesized on the g II If continues Dkazaki lagging strand fragment synthesis A 391 2e 4 Hg A Aquot g a x g r Tram bone mode I 5 The S IUMWFEUT conqm m mc in 4 A assembles a Slldlng Clamp 390 DEBT sliding clamp is oacied quot lt v 39 lt ll mf39 g THE E C l oniotine newiy primed onto the newly prinprpe i ion 9 l g Flaggingstrand re r 7 lt lagging strand 11mp primase 15 j figure 923b o W is released iii ML The trombone model of DNA replication d 591 quot W 339 The secend lagging strand DNA polymerase binds the sliding clamp J at ne primariemp ate junction and Begins to j synthesize a new 39Okezaki fragment The rst agging strand 7 DNA pomymerese is re eased from DN and the ending damp after completion nf an Okezaki fragment Figure QZBde


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