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1、,Chapter33DNAReplication,Outline,GeneralpropertiesofDNAreplicationMajorenzymesandproteinsinvolvedinDNAreplicationDetailedmechanismsofDNAreplication“-form”replicationofgenomicDNAinE.coli“Rolling-circle”replicationD-loopreplicationReplicationofnuclearDNAinEukaryotesArchaealDNAreplicationHighfidelityof
2、DNAReplicationRegulationofDNAreplication,HappyBirthday,DoubleHelix,GeneralFeaturesofDNAReplication,ManyenzymesandproteinsarerequiredTemplatesimilarlyforguanineandcytosine.Thesequenceofbasesonasinglechaindoesnotappeartoberestrictedinanyway.However,ifonlyspecificpairsofbasescanbeformed,itfollowsthatif
3、thesequenceofbasesononechainisgiven,thenthesequenceontheotherchainisautomaticallydetermined.Ithasnotescapedournoticethatthespecificpairingwehavepostulatedimmediatelysuggestsapossiblecopyingmechanismforthegeneticmaterial.Thestructureitselfsuggestedthateachstrandcouldseparateandactasatemplateforanewst
4、rand,thereforedoublingtheamountofDNA,yetkeepingthegeneticinformation,intheformoftheoriginalsequence,intact.,ThreepossiblemodelsforDNAreplication,TestingModelsforDNAreplication,MatthewMeselsonandFranklinStahl(1958),TestingModelsforDNAreplication,DensitylabelingexperimentonE.coliDNA,MeselsonandStahlOr
5、iginalData,SinceDNAreplicationissemi-conservative,thereforethehelixmustbeunwound.JohnCairns(1963)showedthatinitialunwindingislocalizedtoaregionofthebacterialcirculargenome,calledan“origin”or“ori”forshort.,SpecificOriginofReplication,Evidenceofbidirectionalreplication,Eukaryoteshavemanyoriginsofrepli
6、cation,PrimingtheSynthesisofDNA,DNAreplicatesOnlyinthe53direction,ddNTPcanbeusedtoprovethedirectionalityofDNAreplication,SequencepropertiesofDNAreplicationorigins,DNAreplicationissemi-discontinuous,Continuoussynthesis,Discontinuoussynthesis,EnzymesandProteinsInvolvedinDNAReplication,DNAdependentDNAp
7、olymerase(DNApol)-catalyzesincorporationofnucleotidesDNAHelicase-promotesstrandseparation,requiresATPandunwindsdsDNAatreplicationforkSingle-strandedDNAbindingproteins（SSB）-keepstrandsapart,coatDNAandpreventre-associationofstrandsandstimulateDNApolymerasePrimase-catalyzesformationofRNAprimersDNAligas
8、e-joinsOkazakifragmentsTopoisomerase-releasestressofunwinding:relievesstressbybreakingandsealing-otherwiseDNAbecomestootightlycoiledandstopsthereplicatingforkTheEnzymesresponsibleforremovingRNAprimersUracil-DNAN-glycosylase:Removingthemis-incorporateddUMPduringDNAreplicationTelomerase-maintaintelome
9、ricDNAintegrity,DNA-dependentDNApolymerases,CommonReactionEquation:Mg2+DNA+Primer-OH+dNTPDNA/Primer-dNMP+PPi53SubsequenthydrolysisofPPidrivesthereactionforwardBacterialDNApolDNApolI,II,III,IVandVEukaryoticDNApolDNApol,and,AMechanismforAllPolymerases,ThomasA.Steitzhassuggestedthatbiosynthesisofnuclei
10、cacidsproceedsbyanenzymaticmechanismthatisuniversalamongpolymerases.HissuggestionisbasedonstructuralstudiesindicatingthatDNApolymerasesusea“two-metal-ion”mechanismtocatalyzenucleotideadditionduringelongationofagrowingpolynucleotidechain.TheincomingnucleotidehastwoMg2+ionscoordinatedtoitsphosphategro
11、ups,andthesemetalionsinteractwithtwoAspresiduesthatarehighlyconservedinDNA(andRNA)polymerases.Onemetalion,designatedA,interactswiththeOatomofthefree3-OHgrouponthepolynucleotidechain,loweringitsaffinityforitshydrogen.Thisinteractionpromotesnucleophilicattackofthe3-Oonthephosphorusatominthe-phosphateo
12、ftheincomingnucleotide.Thesecondmetalionassistsdepartureoftheproductpyrophosphategroupfromtheincomingnucleotide.Together,thetwometalionsstabilizethepentacovalenttransitionstateonthe-phosphorusatom.,A“two-metal-ion”MechanismforAllPolymerases,E.coliDNApolymerases,IdentificationKornbergandDNApolI(Kornb
13、ergenzyme)StructureandFunctionofDNApolIAmulti-functionalenzymeDNApolIIandDNApolIIIDNApolIVandDNApolVConclusionDNApolIIIisamajorpolymeraseinvolvedinE.colichromosomeDNAreplication,ArthurKornberg(1957),ProteinextractsfromE.coli+TemplateDNAIsnewDNAsynthesized?,dNTPs(substrates)all4atonceMg2+(cofactor)AT
14、P(energysource)free3OHend(primer)InvitroassayforDNAsynthesis,UsedtheassaytopurifyaDNApolymerizingenzymeDNApolI,HowAmazing!,DNAPolIfromE.coliis928aamonomerAsinglepolypeptidewithatleastthreedifferentEnzymaticactivities!a3to5exonucleaseactivitya5to3exonucleaseactivitya5to3DNApolymerizingactivity,Thepro
15、teinisfoldedintodiscretedomains,HansKlenowusedproteases(subtilisinortrypsin)tocleavebetweenresidues323and324,separating5-exonuclease(onthesmallfragment)andtheothertwoactivities(onthelargefragment,theso-calledKlenowfragment”)TomSteitzhasdeterminedthestructureoftheKlenowfragment,MoreonPolI,Whytheexonu
16、cleaseactivity?The3-5exonucleaseactivityservesaproofreadingfunctionItremovesincorrectlymatchedbases,sothatthepolymerasecantryagain,Proofreadingactivityisslowcomparedtopolymerizingactivity,butthestallingofDNAPIafterinsertionofanincorrectbaseallowstheproofreadingactivitytocatchupwiththepolymerizingact
17、ivityandremovetheincorrectbase.,Proofreadingactivityofthe3to5exonuclease,5-exonucleaseactivity,workingtogetherwiththepolymerase,accomplishesnicktranslation,EvenMoreonPolI,In1969JohnCairnsandPauladeLuciaisolatedamutantbacterialstrainwithonly1%DNAPIactivity(polA)mutantwassupersensitivetoUVradiationbut
18、otherwisethemutantwasfineitcoulddivideConclusion:DNAPIisNOTtheprincipalreplicationenzymeinE.coli,DNAPolymeraseIisgreat,but.,DNAPIistooslow(600dNTPsadded/minute)DNAPIisonlymoderatelyprocessive(processivityreferstothenumberofdNTPsaddedtoagrowingDNAchainbeforetheenzymedissociatesfromthetemplate)Conclus
19、ion:TheremustbeadditionalDNApolymerases.BiochemistspurifiedthemfromthepolAmutant,Otherclues.,functionsinmultipleprocessesthatrequireonlyshortlengthsofDNAsynthesishasamajorroleinDNArepair(Cairns-deLuciamutantwasUV-sensitive)itsroleinDNAreplicationistoremoveprimersandfillinthegapsleftbehindforthisitne
20、edsthenick-translationactivity,WhatdoesDNAPIdo?,Atotalof5differentDNAPshavebeenreportedinE.coliDNAPI:does90%ofpolymerizingactivityDNAPII:functionsinDNArepair(provenin1999)DNAPIII:principalDNAreplicationenzymeDNAPIV:functionsinDNArepair(discoveredin1999)DNAPV:functionsinDNArepair(discoveredin1999),Th
21、eDNAPolymeraseFamily,TherealreplicativepolymeraseinE.coliItsfast:upto1,000dNTPsadded/sec/enzymeItshighlyprocessive:500,000dNTPsaddedbeforedissociatingItsaccurate:makes1errorin107dNTPsadded,withproofreading,thisgivesafinalerrorrateof1in1010overall.Geneticmutant(Ts),DNAPolymeraseIII,ITSCOMPLICATED!,Th
22、esubunitsofE.coliDNApolymeraseIII,ThestructureformedbytwobetasubunitsoftheE.coliDNApolymeraseIII.ThisstructurecanclampaDNAmoleculeandslidewiththecorepolymerasealongtheDNAmolecule.,OperationofDNAPolIIIholoenzyme,ComparisonofE.coliDNApolI,II,andIII,EukaryoticDNApolymerase,ActionofHelicase(dnaB),Action
23、ofbacterialSSB,ActionofTopoisomeraseI,ActionofTopoisomeraseII,ActionofDNALigase,The“End-ReplicationProblem”,Theleadingstrandismadeasacontinuousmoleculethatcanreplicateallthewaytotheendofachromosome.ThelaggingstrandismadeasshortOkazakifragments,eachrequiringanewprimertobelaiddownonthetemplate,thatare
24、thenligatedtomakeacontinuousstrand.Thelaggingstrandcannotreplicateallthewaytotheendoflinearchromosome,sincethereisnoDNAbeyondtheendforaprimingeventtofillinthegapbetweenthelastOkazakifragmentandtheterminus.Thisleavesa3overhang.,The“End-ReplicationProblem”anditssolution,Actasprotective“caps”ontheendso
25、fchromosomes.Theyarecomposedofshort,tandemrepeats.Inhumans:5-TTAGGG-3repeatedattheendsofeachchromosomeforatotallengthof15kilobases.Telomeresarenon-codingDNATherefore,iftelomeresgraduallygeterodedbyDNAreplication,thereislessharmtotheorganism,Telomeres,Telomerase=aproteincomponentwithreversetranscript
26、aseactivityplusanRNAcomponentcontaining1.5copiesofthetelomererepeatsequence.ReversetranscriptaseisaDNApolymerasethatusesRNAasatemplate(notDNA)JustlikeotherDNApolymerasesitrequiresaprimer,TelomereRepeatsareAddedbytheenzyme,Telomerase,StructuralmodelofTelomerase,TheRNAcomponentoftelomerasebase-pairswi
27、ththelasttelomererepeat.ThelestofthetelomereRNA“hangsoff”theendofthechromosome.Thismakestheendofthechromosomeintoaprimerthatcanbeextendedbytelomerase.TelomerasemakesaDNAcopyofitsRNA,whichisjustlikeaddingatelomererepeat.Thentheenzymetranslocatesagaintothenewendofthechromosomeandrepeatstheprocess.,How
28、telomeraseworks:,Actionoftelomerase,DetailsofDNAReplication,Threesteps1)Initiation2)Elongation3)TerminationandSeparationDNAreplicationinE.coli-“form”DNAreplicationineukaryotesD-loopreplicationandRolling-circlereplication(-form),ProteinsInvolvedinDNAReplicationinE.coli,DNAReplicationisanOrderedSeries
29、ofSteps,Findtheorigin:DnaA(originrecognitionprotein)+HUUnwindthehelix:DnaB(helicase),DnaC+DnaT(deliverDnaBtotheorigin),SSB(keepshelixunwound),DNAGyrasefacilitatesefficientunwindingSynthesizeprimers:DnaG(primase)+PriA,PriB,PriC(assemblyandfunctionoftheprimosome)Elongate(newstrandsynthesis):DNAPIIIhol
30、oenzymeRemovetheprimersandligateOkazakifragments:(DNAPI+Ligase)Terminatereplication:Ter(terminationsequence)+Tus(terminationutilizationsubstance)SeparateDaughterDNAs:DNATopoIV,Findingandunwindingtheoriginofreplication,13basepairrepeat=5-GATCNTNTTNTT-3,4DnaAtetramersfirstbindtotherepeats.Bindingiscoo
31、perative.EachDnaAbindsATP.,TheyrecruitadditionalDnaAmonomerstobindtoadjacentDNAgeneratinganucleosome-likestructure,DnaApowerstheunwindingofadjacentA-T-richrepeatsbyhydrolyzingATP.AproteincalledHUalsohelps.,DnaB(ahelicase,isnowdeliveredtotheunwoundregionwiththehelpofDnaCandDnaT.Youneedonehelicaseatea
32、chreplicationforktodotheunwinding.DeliveryandassemblyofDnaBontoDNArequiresATP.,SSBcoatstheunwoundDNAstrandstopreventthemfromreassociating.,Unwindingstartsinbothdirections,andshovesoff(displaces)theDnaAproteins.,Thisapreprimingcomplex,PrimaseisnowrecruitedtoeachforksothataprimercanbelaiddownforDNAsyn
33、thesisoneachstrandateachfork.,Primaseisassociatedwithhelicase.PrimaselaysdownanRNAprimerontheleadingstrand.,Primaselaysdownaprimeronthelaggingstrand.,AdditionofDNApolymeraseIIIholoenzymeformsareplisome,PrimersmustbeoccasionallylaiddownonthelaggingstrandtoprimeOkazakifragmentsynthesis.Thisisdonebythe
34、DnaGprimasewhichoccasionallyreassociateswiththeDnaBhelicasetolaydownanewprimeronthelaggingstrand.,Leadingstrand,Leadingstrand,A“snapshot”ofDNAreplication,PolIIIcoredimersynthesizingleadingmanyoftheproteinstakingpartaremoresimilartothoseineukaryoticcellsthantothoseinbacteria.Likebacteria,somearchaeah
35、aveasinglereplicationorigin,butthearchaeanSulfolobussolfataricushastwooriginsofreplication,similartothemultipleoriginsseenineukaryoticgenomes.Thereplicationoriginsofarchaeadonotcontainthetypicalsequencesrecognizedbybacterialinitiatorproteins;instead,theyhavesequencesthataresimilartothosefoundineukar
36、yoticorigins.Theinitiatorproteinsofarchaeaalsoaremoresimilartothoseofeukaryotesthanthoseofbacteria.Thesesimilaritiesinreplicationbetweenarchaealandeukaryoticcellsreinforcetheconclusionthatthearchaeaaremorecloselyrelatedtoeukaryoticcellsthantotheprokaryoticbacteria.,HighFidelityofReplication,Balanced
37、levelsofdNTPs.HighselectivityofDNAPsbasedonWatson-Crickbasepairing(tothetemplatebase)ProofreadingofDNAPsbymeansoftheir3-5exonuclease.Mismatchrepairsystem.RNAprimersareremovedbyhighlyaccuratePolIenzyme.,DNApolymeraseerrorrates,Initialpairingerror=1/105Afterproofreading=1/1071/108mismatchrepair=1/10101/1011Humangenome=3.2x109bp3errors/replication!,APoem(Englishversion),Replicativeerrorsarenobigdeal,Aslongaseditingisright.Evenoccursonemistake,Repairingenzymeswillcorrectitlater.,RongwuYang(InspiredbyDNAreplication),