DNA-replication@生物化学讲义课件

HappyBirthday,

DoubleHelix

HappyBirthday,

DoubleHelixDNAReplicationBackgroundInformationWatson&CrickGeneralFeatures1)Manyenzymesandproteinsarerequired2)Template&dNTPs/Mg2+arerequired3)Semi-conservativeAkeyexperimentdesignedbyM.MeselsonandW.F.Stahl（1958）4)DNAUnwindingisnecessary5)APrimerwithafree3'-OHgroupisrequired6)Onlyinthe5′→3′direction7)SpecificOriginofReplication-OriCandARS(AutonomouslyReplicatingSequence）

ThreeCommonFeaturesofReplicationOrigins

8)Bi-directional(Withsomeexceptions)9)Semi-discontinuous

Replicationfork，Leadingstrand,Laggingstrand

andOkazakifragments10)Highlyprocessive,HighlyorderedandExtremelyaccurateDNAReplicationBackgroundInfo2"MolecularStructureofNucleicAcids:

AStructureforDeoxyriboseNucleicAcid"

(Nature,April25,1953.volume171:737-738.)

"Thenovelfeatureofthestructureisthemannerinwhichthetwochainsareheldtogetherbythepurineandpyrimidinebases...The(bases)arejoinedtogetherinpairs,asinglebasefromonechainbeinghydrogen-bondedtoasinglebasefromtheotherchain,sothatthetwoliesidebyside...Oneofthepairmustbeapurineandtheotherapyrimidineforbondingtooccur....Onlyspecificpairsofbasescanbondtogether.Thesepairsare:adenine(purine)withthymine(pyrimidine),andguanine(purine)withcytosine(pyrimidine).""...inotherwords,ifanadenineformsonememberofapair,oneitherchain,thenontheseassumptionstheothermembermustbethymine;similarlyforguanineandcytosine.Thesequenceofbasesonasinglechaindoesnotappeartoberestrictedinanyway.However,ifonlyspecificpairsofbasescanbeformed,itfollowsthatifthesequenceofbasesononechainisgiven,thenthesequenceontheotherchainisautomaticallydetermined.""...Ithasnotescapedournoticethatthespecificpairingwehavepostulatedimmediatelysuggestsapossiblecopyingmechanismforthegeneticmaterial.Thestructureitselfsuggestedthateachstrandcouldseparateandactasatemplateforanewstrand,thereforedoublingtheamountofDNA,yetkeepingthegeneticinformation,intheformoftheoriginalsequence,intact.""MolecularStructureofNuclei3DNA-replication@生物化学讲义课件TestingModelsforDNAreplicationMatthewMeselsonandFranklinStahl(1958)TestingModelsforDNAreplicaMatthewMeselsonandFranklinStahlmorerecentlyFacultymemberatHarvardMechanismsofMolecularEvolutionFacultyChairforCBWStudiesFacultymemberatU.ofOregonMeioticRecombinationMatthewMeselsonandFranklinTestingModelsforDNAreplicationMeselsonandStahl(1958)DensitylabelingexperimentonE.coli(bacterial)DNABacterialculture15NH4Cl(SoleNsource)GrowforseveralgenerationsBacterialculturewithdenseDNAThisisthestartingmaterialfortheexperimentTestingModelsforDNAreplicaMeselsonandStahl(continued)Harvestcellsandresuspendinmediawith14NH4Cl

asthesoleNsourceBacterialculturewithdenseDNAGrowfor1generationHarvestsomecells“1stgeneration”GrowforanothergenerationHarvestsomecells“2ndgeneration”GrowforanothergenerationetcForeachgenerationisolatetheDNAandspinthroughadensity(CsCl)gradient).DetectDNAinthegradient(egbyUVabsorption)MonitorhowmanyDNAbandsthereareaftereachgenerationBacterialculture“0generation”14NH4ClMeselsonandStahl(continued)DNA-replication@生物化学讲义课件MeselsonandStahlOriginalDataMeselsonandStahlOriginalDaDNA-replication@生物化学讲义课件DNAReplicationSinceDNAreplicationissemiconservative,thereforethehelixmustbeunwound.JohnCairns(1963)showedthatinitialunwindingislocalizedtoaregionofthebacterialcirculargenome,calledan“origin”or“ori”forshort.DNAReplicationSinceDNArepl12E.colichromosomeLocalizedunwindingoriginORDNAreplicationunidirectionalbidirectionalReplicationforksE.coliLocalizedoriginORDNAreJohnCairnsGrowcellsforseveralgenerationsSmallamountsof3HthymidineareincorporatedintonewDNAGrowforbriefperiodoftimeAddahighconcentrationof3H-thymidineinmediawithlowconcentrationof3H-thymidineBacterialculture*T*T*T*TDenselabelatthereplicationforkwherenewDNAisbeingmade*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*TAllDNAislightlylabeledwithradioactivity*T*T*TCairnsthenisolatedthechromosomesbylysingthecellsveryverygentlyandplacedthemonanelectronmicrograph(EM)gridwhichheexposedtoX-rayfilmfortwomonths.JohnCairnsGrowcellsforseveEvidencepointstobidirectionalreplicationLabelatbothreplicationforksEvidencepointstobidirectionDNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNAReplicationisSemi-discontinuousConsideronereplicationfork:5’3’5’3’DirectionofunwindingContinuousreplication5’3’PrimerPrimer5’3’Primer5’3’DiscontinuousreplicationDNAReplicationisSemi-disconEvidencefortheSemi-DiscontinuousreplicationmodelwasprovidedbytheOkazakis(1968)ReijiOkazakiwasbornnearHiroshima,Japan,in1930.HewasateenagerthereatthetimeoftheexplosionofthefirstoftwonuclearbombsthattheUSdroppedattheendofWorldWarII.Hisscientificcareerwascutshortbyhisuntimelydeathfromcancerin1975attheageof44,perhapsrelatedtohisexposuretothefalloutofthatblast.EvidencefortheSemi-DiscontiEvidenceforSemi-DiscontinuousReplication(pulse-chaseexperiment)BacteriaarereplicatingBacterialcultureAdd3HThymidineForaSHORTtime(i.e.seconds)Floodwithnon-radioactiveTAllowreplicationTocontinueHarvestthebacteriaatdifferenttimesafterthechaseIsolatetheirDNASeparatethestrands(usingalkaliconditions)RunonasizinggradientsmallestlargestRadioactivitywillonlybeintheDNAthatwasmadeduringthepulseEvidenceforSemi-DiscontinuousmallestlargestResultsofpulse-chaseexperimentPulse5’3’5’3’Directionofunwinding3’5’PrimerPrimer5’3’Primer5’3’******ChasesmallestlargestResultsofpulsContinuoussynthesisDiscontinuoussynthesisDNAreplicationissemi-discontinuousContinuoussynthesisDiscontinuEnzymesandProteinsInvolvedinDNAReplicationDNA–dependentDNApolymerase(DNApol,DNA聚合酶）-incorporationofnucleotidesDNAHelicase（DNA解链酶）-promotes

strandseparation,requiresATPandunwindsdsDNAatreplicationfork

Single-strandedDNAbindingproteins（

SSB，单链结合蛋白）-keepstrandsapart,

coatDNAandpreventre-associationofstrandsandstimulateDNApolymerase

Primase（引发酶）-

formationofRNAprimersDNAligase

（DNA连接酶）-joiningofOkazakifragmentsTopoisomerase（拓扑异构酶）-releasestressofunwinding:

relievesstressbybreakingandsealing--otherwiseDNAbecomestootightlycoiledandstopsthereplicatingfork

TheEnzymesresponsibleforremovingRNAprimers

Uracil-DNAN-glycosylase

（尿嘧啶-DNA-N-糖苷酶）Telomerase（端聚酶）-maintaintelomericDNAintegrityEnzymesandProteinsInvolved29DNA-dependentDNApolymerasesCommonReactionEquation:

Mg2+

DNA+Primer-OH+dNTPDNA/Primer-dNMP+PPi

5′3′

Subsequent

hydrolysisof

PPidrivesthe

reactionforwardProkaryoticDNApol

DNApolI,II,III,IVandVEukaryoticDNApol

DNApolα,β,γ,δandεDNA-dependentDNApolymerasesC30E.coliDNApolymerasesIdentification

KornbergandDNApolI(Kornbergenzyme)StructureandFunctionofDNApolI

Amulti-functionalenzymeDNApolIIandDNApolIIIDNApolIVandDNApolVConclusion

DNApolIIIisamajorpolymeraseinvolvedinE.colichromosomeDNAreplicationE.coliDNApolymerasesIdentif31ArthurKornberg(1957)ProteinextractsfromE.coli+TemplateDNAIsnewDNAsynthesized??-dNTPs(substrates)all4atonce-Mg2+(cofactor)-ATP(energysource)-free3’OHend(primer)InvitroassayforDNAsynthesisUsedtheassaytopurifyaDNApolymerizingenzymeDNApolymeraseICurrentlyafacultymemberatStanfordSchoolofMedicineArthurKornberg(1957)ProteinHowAmazing!!!a3’to5’exonucleaseactivitya5’to3’exonucleaseactivitya5’to3’DNApolymerizingactivityDNAPolIfromE.coliis928aa(109kD)monomerAsinglepolypeptidewithatleastthreedifferentEnzymaticactivities!HowAmazing!!!DNATheproteinisfoldedintodiscretedomainsHansKlenowusedproteases(subtilisinortrypsin)tocleavebetweenresidues323and324,separating5'-exonuclease(onthesmallfragment)andtheothertwoactivities(onthelargefragment,theso-called"Klenowfragment”)TomSteitzhasdeterminedthestructureoftheKlenowfragmentTheproteinisfoldedintodisMoreonPolI

Whytheexonucleaseactivity?

The3'-5'exonucleaseactivityservesaproofreadingfunctionItremovesincorrectlymatchedbases,sothatthepolymerasecantryagainMoreonPolIWhytheexonucle35ConceptualmodelforproofreadingbasedonkineticconsiderationsstallingtransientmeltingexonucleasesiteoccupancyConceptualmodelforproofread36Proofreadingactivityofthe3’to5’exonuclease.Proofreadingactivityisslowcomparedtopolymerizingactivity,butthestallingofDNAPIafterinsertionofanincorrectbaseallowstheproofreadingactivitytocatchupwiththepolymerizingactivityandremovetheincorrectbase.ProofreadingactivityNoticehowthenewly-formedstrandoscillatesbetweenthepolymeraseand3'-exonucleasesites,addingabaseandthencheckingitMoreonPolI3’to5’exonucleaseactivity

Noticehowthenewly-formedstStructureoftheKlenowfragmentStructureoftheKlenowfragmeDNA-replication@生物化学讲义课件EvenMoreonPolI

5'-exonucleaseactivity,workingtogetherwiththepolymerase,accomplishes"nicktranslation"EvenMoreonPolI5'-exonucle41DNAPolymeraseIisgreat,but….

In1969JohnCairnsandPauladeLucia

-isolatedamutantbacterialstrainwithonly1%DNAPIactivity(polA)-mutantwassupersensitivetoUVradiation-butotherwisethemutantwasfine-itcoulddivideConclusion:

DNAPIisNOTtheprincipalreplicationenzymeinE.coliDNAPolymeraseIisgreat,but42Otherclues….

-DNAPIistooslow(600dNTPsadded/minute)-DNAPIisonlymoderatelyprocessive (processivityreferstothenumberofdNTPsaddedtoagrowingDNAchainbeforetheenzymedissociatesfromthetemplate)Conclusion:

TheremustbeadditionalDNApolymerases.BiochemistspurifiedthemfromthepolAmutantOtherclues….-DNAPIistoo43WhatdoesDNAPIdo?

-functionsinmultipleprocessesthatrequireonlyshortlengthsofDNAsynthesis-hasamajorroleinDNArepair(Cairns-deLuciamutantwasUV-sensitive)-itsroleinDNAreplicationistoremoveprimersandfillinthegapsleftbehind-forthisitneedsthenick-translationactivityWhatdoesDNAPIdo?-functio44TheDNAPolymeraseFamily

Atotalof5differentDNAPshavebeenreportedinE.coli

DNAPI:does90%ofpolymerizingactivity

DNAPII:functionsinDNArepair

(provenin1999)DNAPIII:principalDNAreplicationenzyme

DNAPIV:functionsinDNArepair(discoveredin1999)DNAPV:functionsinDNArepair(discoveredin1999)

TheDNAPolymeraseFamilyAto45DNAPolymeraseIIIThe"real"replicativepolymeraseinE.coli

It’sfast:upto1,000dNTPsadded/sec/enzymeIt’shighlyprocessive:>500,000dNTPsaddedbeforedissociatingIt’saccurate:makes1errorin107dNTPsadded,withproofreading,thisgivesafinalerrorrateof1in1010overall.Geneticmutant(Ts)IT’SCOMPLICATED!!!DNAPolymeraseIIIThe"real"46ThesubunitsofE.coliDNApolymeraseIIISubunitFunctionaeqtbgdd’cy5’to3’polymerizingactivity3’to5’exonucleaseactivityaandeassembly(scaffold)AssemblyofholoenzymeonDNASlidingclamp=processivityfactorClamp-loadingcomplexClamp-loadingcomplexClamp-loadingcomplexClamp-loadingcomplexClamp-loadingcomplexCoreEnzymedimerHoloenzymeThesubunitsofE.coliDNApoThestructureformedbytwobetasubunitsof

theE.coliDNApolymeraseIII.

ThisstructurecanclampaDNAmoleculeandslidewiththecorepolymerasealongtheDNAmolecule.Thestructureformedbytwobe48DNAPolymeraseIII

holoenzymeCoreCorebbbbbclampst2tsubunitshold2coresinadimergcomplex(clamploader)gReplicationForkLeadingStrandsynthesisLaggingStrandsynthesisDNAPolymeraseIII

holoenzyme49ComparisonofE.Coli

DNApolI,II,andIIIComparisonofE.Coli

DNApol50EukaryoticDNApolymeraseEukaryoticDNApolymerase51OtherEnzymesandProteinsInvolvedinDNAReplicationHelicase:IandII；ATPaseHelicaseIIisinvolvedinDNAreplicationE.coli:dnaB蛋白andRep蛋白

Wernersyndrome(WS)andHelicasemutationSSB：withoutanyenzymaticactivity

Prokaryotic:ActinacooperativefashionEukaryotic:ReplicationFactorA(RFA)Primase:AkindofDNA-dependentRNApolymeraseTheEnzymeremovingprimers

Prokaryotic:DNApolI;Enkaryotic:RNaseH(5’-3’exonucleaseactivityactiveonlyonRNA-DNAhybrids)

orMF1(5’-3’exonuclease)DNAligase

Prokaryotic:NAD+;EukaryoticandViral:ATPTopoisomerase:I,II(E.coli-Gyrase),III,andIVIIandIVareinvolvedinDNAreplicationUracil-DNAN-glycosylaseRemovingthemis-incorporateddUMPduringDNAreplicationTelomease

Specifictoeukaryotes;Akindofretro-transcriptaseOtherEnzymesandProteinsInv52DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件DNA-replication@生物化学讲义课件ActionofTopoisomeraseIIActionofTopoisomeraseII56ActionofDNALigaseActionofDNALigase57DNA-replication@生物化学讲义课件The“End-ReplicationProblem”Theleadingstrandismadeasacontinuousmoleculethatcanreplicateallthewaytotheendofachromosome.ThelaggingstrandismadeasshortOkazakifragments,eachrequiringanewprimertobelaiddownonthetemplate,thatarethenligatedtomakeacontinuousstrand.Thelaggingstrandcannotreplicateallthewaytotheendoflinearchromosome,sincethereisnoDNAbeyondtheendforaprimingeventtofillinthegapbetweenthelastOkazakifragmentandtheterminus.Thisleavesa3’overhang.The“End-ReplicationProblem”TActasprotective“caps”ontheendsofchromosomes.Theyarecomposedofshort,tandemrepeats.Inhumans:5’-TTAGGG-3’repeatedattheendsofeachchromosomeforatotallengthof15kilobases.Telomeresarenon-codingDNATherefore,iftelomeresgraduallygeterodedbyDNAreplication,thereislessharmtotheorganismTelomeresActasprotective“caps”onthTelomerase=aproteincomponentwithreversetranscriptaseactivityplusanRNAcomponentcontaining1.5copiesofthetelomererepeatsequence.ReversetranscriptaseisaDNApolymerasethatusesRNAasatemplate(notDNA)JustlikeotherDNApolymerasesitrequiresaprimerTelomereRepeatsareAddedbytheenzyme,TelomeraseTelomerase=aproteincomponeDNA-replication@生物化学讲义课件TheRNAcomponentoftelomerasebase-pairswiththelasttelomererepeat.ThelestofthetelomereRNA“hangsoff”theendofthechromosome.Thismakestheendofthechromosomeintoaprimerthatcanbeextendedbytelomerase.TelomerasemakesaDNAcopyofitsRNA,whichisjustlikeaddingatelomererepeat.Thentheenzymetranslocatesagaintothenewendofthechromosomeandrepeatstheprocess.Howtelomeraseworks:TheRNAcomponentoftelomerasDNA-replication@生物化学讲义课件DetailsofDNAReplicationThreesteps

1)Initiation（起始）

2)Elongation（延伸）

3)TerminationandSeparation（终止与分离）DNAreplicationinE.coli-“θform”DNAreplicationineukaryotesD-loopreplicationandRolling-circlereplication(σ-form)DetailsofDNAReplicationThre65ProteinsInvolvedinDNAReplicationinE.coliProteinsInvolvedinDNARepliDNAReplicationisanOrderedSeriesofStepsFindtheorigin:DnaA(originrecognitionprotein)+HUUnwindthehelix:DnaB(helicase),DnaC+DnaT(deliverDnaB

totheorigin),SSB(keepshelixunwound),DNAGyrase

facilitatesefficientunwindingSynthesizeprimers:DnaG(primase)+PriA,PriB,PriC

(assemblyandfunctionoftheprimosome)Elongate

(newstrandsynthesis):DNAPIIIholoenzymeRemovetheprimersandligate

Okazakifragments:(DNAPI+Ligase)Terminatereplication:Ter(terminationsequence)+Tus(terminationutilizationsubstance)

SeparateDaughterDNAs:DNATopoIVPrimosome-引发体Gyrase-旋转酶DNAReplicationisanOrderedFindingandunwindingtheoriginofreplication13basepairrepeat=5’-GATCNTNTTNTT-3’4DnaAtetramersfirstbindtotherepeats.Bindingiscooperative.EachDnaAbindsATP.TheyrecruitadditionalDnaAmonomerstobindtoadjacentDNAgeneratinganucleosome-likestructureDnaApowerstheunwindingofadjacentA-T-richrepeatsbyhydrolyzingATP.AproteincalledHUalsohelps.FindingandunwindingtheorigDnaB(ahelicase,isnowdeliveredtotheunwoundregionwiththehelpofDnaCandDnaT.Youneedonehelicaseateachreplicationforktodotheunwinding.DeliveryandassemblyofDnaBontoDNArequiresATP.SSBcoatstheunwoundDNAstrandstopreventthemfromreassociating.Unwindingstartsinbothdirections,andshovesoff(displaces)theDnaAproteins.Thisapreprimingcomplex.

DnaB(ahelicase,isnowdeliPrimaseisnowrecruitedtoeachforksothataprimercanbelaiddownforDNAsynthesisoneachstrandateachfork.Primaseisassociatedwithhelicase.PrimaselaysdownanRNAprimerontheleadingstrand.Primaselaysdownaprimeronthelaggingstrand.Thisaprimosome.

PrimaseisnowrecruitedtoeaAdditionofDNApolymeraseIIIholoenzymeformsareplisomePrimersmustbeoccasionallylaiddownonthelaggingstrandtoprimeOkazakifragmentsynthesis.ThisisdonebytheDnaGprimasewhichoccasionallyreassociateswiththeDnaBhelicasetolaydownanewprimeronthelaggingstrand.LeadingstrandLeadingstrandAdditionofDNApolymeraseIIIA“snapshot”ofDNAreplicationA“snapshot”ofDNAreplicatioPolIIIcoredimersynthesizingleading&laggingstrands.TausubunitofPolIIIbindstohelicase.PolIIIcoredimersynthesizinbClamploader

gComplexofPolIIIholoenzyme(g2,d,d’,c,psi)

UsesATPtoopenβdimerandpositionitat3’-endofprimer.“Loaded”βclampthenbindsPolIIIcore(and releasesfromγ).ProcessiveDNAsynthesis.-loadsbsubunitdimerontoprimerOrderofeventsbClamploadergComplexofPoRecyclingphaseOnceOkazakifragmentcompleted,bclampreleasesfromcore.

bbindstog.

g

unloadsbclampfromDNA.

bclamprecyclestonextprimer.RecyclingphaseOnceOkazakifr75

TerminationofReplicationTerminationoccursatterregionofE.colichromosome.terregionrichinGsandTs,signalstheendofreplication.Terminatorutilizationsubstance(Tus)bindstoterregion.Tuspreventsreplicationforkfrompassingbyinhibitinghelicaseactivity.TerminationofReplicationTerm77DNA-replication@生物化学讲义课件TerminatingDNAsynthesisinprokaryotes.Fig.21.27EachforkstopsattheTerregions,whichare22bp,3copies,andbindtheTusprotein.TerminatingDNAsynthesisinpDNA-replication@生物化学讲义课件EukaryoticDNA

Replication

LikeE.coli,butmorecomplex

ChromatinandNucleosomeMultipleoriginsofreplication

DNAreplicationoccursjustatSphaseofthecellcycleandiscontrolledbymanyproteinsOkazakifragmentsareshorterthaninProkaryotesReplicationforksrunaslowerspeedthaninProkaryotesTworoundsofreplicationcannotoccuratthesametimeTelomeraseisrequiredEukaryoticDNAReplicationLik81DNA-replication@生物化学讲义课件Licensing:positivecontrolofEukaryoticDNAreplication

AnOriginRecognitionComplexofproteins(ORC).TheseremainontheDNAthroughouttheprocess.Accessoryproteinscalledlicensingfactors.TheseaccumulateinthenucleusduringG1ofthecellcycle.Theyinclude:

Cdc6andCdt1,whichbindtotheORCandareessentialforcoatingtheDNAwith

MCMproteins.OnlyDNAcoatedwithMCMproteins(thereare6ofthem)canbereplicated.Oncereplicat