Chapter33 DNA replication.ppt

,Chapter33DNAReplication,Outline,GeneralpropertiesofDNAreplicationMajorenzymesandproteinsinvolvedinDNAreplicationDetailedmechanismsofDNAreplication“-form”replicationofgenomicDNAinE.coli“Rolling-circle”replicationD-loopreplicationReplicationofnuclearDNAinEukaryotesArchaealDNAreplicationHighfidelityofDNAReplicationRegulationofDNAreplication,HappyBirthday,DoubleHelix,GeneralFeaturesofDNAReplication,ManyenzymesandproteinsarerequiredTemplatesimilarlyforguanineandcytosine.Thesequenceofbasesonasinglechaindoesnotappeartoberestrictedinanyway.However,ifonlyspecificpairsofbasescanbeformed,itfollowsthatifthesequenceofbasesononechainisgiven,thenthesequenceontheotherchainisautomaticallydetermined."".Ithasnotescapedournoticethatthespecificpairingwehavepostulatedimmediatelysuggestsapossiblecopyingmechanismforthegeneticmaterial.Thestructureitselfsuggestedthateachstrandcouldseparateandactasatemplateforanewstrand,thereforedoublingtheamountofDNA,yetkeepingthegeneticinformation,intheformoftheoriginalsequence,intact.",ThreepossiblemodelsforDNAreplication,TestingModelsforDNAreplication,MatthewMeselsonandFranklinStahl(1958),TestingModelsforDNAreplication,DensitylabelingexperimentonE.coliDNA,MeselsonandStahlOriginalData,SinceDNAreplicationissemi-conservative,thereforethehelixmustbeunwound.JohnCairns(1963)showedthatinitialunwindingislocalizedtoaregionofthebacterialcirculargenome,calledan“origin”or“ori”forshort.,SpecificOriginofReplication,Evidenceofbidirectionalreplication,Eukaryoteshavemanyoriginsofreplication,PrimingtheSynthesisofDNA,DNAreplicatesOnlyinthe53direction,ddNTPcanbeusedtoprovethedirectionalityofDNAreplication,SequencepropertiesofDNAreplicationorigins,DNAreplicationissemi-discontinuous,Continuoussynthesis,Discontinuoussynthesis,EnzymesandProteinsInvolvedinDNAReplication,DNAdependentDNApolymerase(DNApol)-catalyzesincorporationofnucleotidesDNAHelicase-promotesstrandseparation,requiresATPandunwindsdsDNAatreplicationforkSingle-strandedDNAbindingproteins（SSB）-keepstrandsapart,coatDNAandpreventre-associationofstrandsandstimulateDNApolymerasePrimase-catalyzesformationofRNAprimersDNAligase-joinsOkazakifragmentsTopoisomerase-releasestressofunwinding:relievesstressbybreakingandsealing-otherwiseDNAbecomestootightlycoiledandstopsthereplicatingforkTheEnzymesresponsibleforremovingRNAprimersUracil-DNAN-glycosylase:Removingthemis-incorporateddUMPduringDNAreplicationTelomerase-maintaintelomericDNAintegrity,DNA-dependentDNApolymerases,CommonReactionEquation:Mg2+DNA+Primer-OH+dNTPDNA/Primer-dNMP+PPi53SubsequenthydrolysisofPPidrivesthereactionforwardBacterialDNApolDNApolI,II,III,IVandVEukaryoticDNApolDNApol,and,AMechanismforAllPolymerases,ThomasA.Steitzhassuggestedthatbiosynthesisofnucleicacidsproceedsbyanenzymaticmechanismthatisuniversalamongpolymerases.HissuggestionisbasedonstructuralstudiesindicatingthatDNApolymerasesusea“two-metal-ion”mechanismtocatalyzenucleotideadditionduringelongationofagrowingpolynucleotidechain.TheincomingnucleotidehastwoMg2+ionscoordinatedtoitsphosphategroups,andthesemetalionsinteractwithtwoAspresiduesthatarehighlyconservedinDNA(andRNA)polymerases.Onemetalion,designatedA,interactswiththeOatomofthefree3-OHgrouponthepolynucleotidechain,loweringitsaffinityforitshydrogen.Thisinteractionpromotesnucleophilicattackofthe3-Oonthephosphorusatominthe-phosphateoftheincomingnucleotide.Thesecondmetalionassistsdepartureoftheproductpyrophosphategroupfromtheincomingnucleotide.Together,thetwometalionsstabilizethepentacovalenttransitionstateonthe-phosphorusatom.,A“two-metal-ion”MechanismforAllPolymerases,E.coliDNApolymerases,IdentificationKornbergandDNApolI(Kornbergenzyme)StructureandFunctionofDNApolIAmulti-functionalenzymeDNApolIIandDNApolIIIDNApolIVandDNApolVConclusionDNApolIIIisamajorpolymeraseinvolvedinE.colichromosomeDNAreplication,ArthurKornberg(1957),ProteinextractsfromE.coli+TemplateDNAIsnewDNAsynthesized?,dNTPs(substrates)all4atonceMg2+(cofactor)ATP(energysource)free3OHend(primer)InvitroassayforDNAsynthesis,UsedtheassaytopurifyaDNApolymerizingenzymeDNApolI,HowAmazing!,DNAPolIfromE.coliis928aamonomerAsinglepolypeptidewithatleastthreedifferentEnzymaticactivities!a3to5exonucleaseactivitya5to3exonucleaseactivitya5to3DNApolymerizingactivity,Theproteinisfoldedintodiscretedomains,HansKlenowusedproteases(subtilisinortrypsin)tocleavebetweenresidues323and324,separating5-exonuclease(onthesmallfragment)andtheothertwoactivities(onthelargefragment,theso-called"Klenowfragment”)TomSteitzhasdeterminedthestructureoftheKlenowfragment,MoreonPolI,Whytheexonucleaseactivity?The3-5exonucleaseactivityservesaproofreadingfunctionItremovesincorrectlymatchedbases,sothatthepolymerasecantryagain,Proofreadingactivityisslowcomparedtopolymerizingactivity,butthestallingofDNAPIafterinsertionofanincorrectbaseallowstheproofreadingactivitytocatchupwiththepolymerizingactivityandremovetheincorrectbase.,Proofreadingactivityofthe3to5exonuclease,5-exonucleaseactivity,workingtogetherwiththepolymerase,accomplishes"nicktranslation",EvenMoreonPolI,In1969JohnCairnsandPauladeLuciaisolatedamutantbacterialstrainwithonly1%DNAPIactivity(polA)mutantwassupersensitivetoUVradiationbutotherwisethemutantwasfineitcoulddivideConclusion:DNAPIisNOTtheprincipalreplicationenzymeinE.coli,DNAPolymeraseIisgreat,but.,DNAPIistooslow(600dNTPsadded/minute)DNAPIisonlymoderatelyprocessive(processivityreferstothenumberofdNTPsaddedtoagrowingDNAchainbeforetheenzymedissociatesfromthetemplate)Conclusion:TheremustbeadditionalDNApolymerases.BiochemistspurifiedthemfromthepolAmutant,Otherclues.,functionsinmultipleprocessesthatrequireonlyshortlengthsofDNAsynthesishasamajorroleinDNArepair(Cairns-deLuciamutantwasUV-sensitive)itsroleinDNAreplicationistoremoveprimersandfillinthegapsleftbehindforthisitneedsthenick-translationactivity,WhatdoesDNAPIdo?,Atotalof5differentDNAPshavebeenreportedinE.coliDNAPI:does90%ofpolymerizingactivityDNAPII:functionsinDNArepair(provenin1999)DNAPIII:principalDNAreplicationenzymeDNAPIV:functionsinDNArepair(discoveredin1999)DNAPV:functionsinDNArepair(discoveredin1999),TheDNAPolymeraseFamily,The"real"replicativepolymeraseinE.coliItsfast:upto1,000dNTPsadded/sec/enzymeItshighlyprocessive:>500,000dNTPsaddedbeforedissociatingItsaccurate:makes1errorin107dNTPsadded,withproofreading,thisgivesafinalerrorrateof1in1010overall.Geneticmutant(Ts),DNAPolymeraseIII,ITSCOMPLICATED!,ThesubunitsofE.coliDNApolymeraseIII,ThestructureformedbytwobetasubunitsoftheE.coliDNApolymeraseIII.ThisstructurecanclampaDNAmoleculeandslidewiththecorepolymerasealongtheDNAmolecule.,OperationofDNAPolIIIholoenzyme,ComparisonofE.coliDNApolI,II,andIII,EukaryoticDNApolymerase,ActionofHelicase(dnaB),ActionofbacterialSSB,ActionofTopoisomeraseI,ActionofTopoisomeraseII,ActionofDNALigase,The“End-ReplicationProblem”,Theleadingstrandismadeasacontinuousmoleculethatcanreplicateallthewaytotheendofachromosome.ThelaggingstrandismadeasshortOkazakifragments,eachrequiringanewprimertobelaiddownonthetemplate,thatarethenligatedtomakeacontinuousstrand.Thelaggingstrandcannotreplicateallthewaytotheendoflinearchromosome,sincethereisnoDNAbeyondtheendforaprimingeventtofillinthegapbetweenthelastOkazakifragmentandtheterminus.Thisleavesa3overhang.,The“End-ReplicationProblem”anditssolution,Actasprotective“caps”ontheendsofchromosomes.Theyarecomposedofshort,tandemrepeats.Inhumans:5-TTAGGG-3repeatedattheendsofeachchromosomeforatotallengthof15kilobases.Telomeresarenon-codingDNATherefore,iftelomeresgraduallygeterodedbyDNAreplication,thereislessharmtotheorganism,Telomeres,Telomerase=aproteincomponentwithreversetranscriptaseactivityplusanRNAcomponentcontaining1.5copiesofthetelomererepeatsequence.ReversetranscriptaseisaDNApolymerasethatusesRNAasatemplate(notDNA)JustlikeotherDNApolymerasesitrequiresaprimer,TelomereRepeatsareAddedbytheenzyme,Telomerase,StructuralmodelofTelomerase,TheRNAcomponentoftelomerasebase-pairswiththelasttelomererepeat.ThelestofthetelomereRNA“hangsoff”theendofthechromosome.Thismakestheendofthechromosomeintoaprimerthatcanbeextendedbytelomerase.TelomerasemakesaDNAcopyofitsRNA,whichisjustlikeaddingatelomererepeat.Thentheenzymetranslocatesagaintothenewendofthechromosomeandrepeatstheprocess.,Howtelomeraseworks:,Actionoftelomerase,DetailsofDNAReplication,Threesteps1)Initiation2)Elongation3)TerminationandSeparationDNAreplicationinE.coli-“form”DNAreplicationineukaryotesD-loopreplicationandRolling-circlereplication(-form),ProteinsInvolvedinDNAReplicationinE.coli,DNAReplicationisanOrderedSeriesofSteps,Findtheorigin:DnaA(originrecognitionprotein)+HUUnwindthehelix:DnaB(helicase),DnaC+DnaT(deliverDnaBtotheorigin),SSB(keepshelixunwound),DNAGyrasefacilitatesefficientunwindingSynthesizeprimers:DnaG(primase)+PriA,PriB,PriC(assemblyandfunctionoftheprimosome)Elongate(newstrandsynthesis):DNAPIIIholoenzymeRemovetheprimersandligateOkazakifragments:(DNAPI+Ligase)Terminatereplication:Ter(terminationsequence)+Tus(terminationutilizationsubstance)SeparateDaughterDNAs:DNATopoIV,Findingandunwindingtheoriginofreplication,13basepairrepeat=5-GATCNTNTTNTT-3,4DnaAtetramersfirstbindtotherepeats.Bindingiscooperative.EachDnaAbindsATP.,TheyrecruitadditionalDnaAmonomerstobindtoadjacentDNAgeneratinganucleosome-likestructure,DnaApowerstheunwindingofadjacentA-T-richrepeatsbyhydrolyzingATP.AproteincalledHUalsohelps.,DnaB(ahelicase,isnowdeliveredtotheunwoundregionwiththehelpofDnaCandDnaT.Youneedonehelicaseateachreplicationforktodotheunwinding.DeliveryandassemblyofDnaBontoDNArequiresATP.,SSBcoatstheunwoundDNAstrandstopreventthemfromreassociating.,Unwindingstartsinbothdirections,andshovesoff(displaces)theDnaAproteins.,Thisapreprimingcomplex,PrimaseisnowrecruitedtoeachforksothataprimercanbelaiddownforDNAsynthesisoneachstrandateachfork.,Primaseisassociatedwithhelicase.PrimaselaysdownanRNAprimerontheleadingstrand.,Primaselaysdownaprimeronthelaggingstrand.,AdditionofDNApolymeraseIIIholoenzymeformsareplisome,PrimersmustbeoccasionallylaiddownonthelaggingstrandtoprimeOkazakifragmentsynthesis.ThisisdonebytheDnaGprimasewhichoccasionallyreassociateswiththeDnaBhelicasetolaydownanewprimeronthelaggingstrand.,Leadingstrand,Leadingstrand,A“snapshot”ofDNAreplication,PolIIIcoredimersynthesizingleadingmanyoftheproteinstakingpartaremoresimilartothoseineukaryoticcellsthantothoseinbacteria.Likebacteria,somearchaeahaveasinglereplicationorigin,butthearchaeanSulfolobussolfataricushastwooriginsofreplication,similartothemultipleoriginsseenineukaryoticgenomes.Thereplicationoriginsofarchaeadonotcontainthetypicalsequencesrecognizedbybacterialinitiatorproteins;instead,theyhavesequencesthataresimilartothosefoundineukaryoticorigins.Theinitiatorproteinsofarchaeaalsoaremoresimilartothoseofeukaryotesthanthoseofbacteria.Thesesimilaritiesinreplicationbetweenarchaealandeukaryoticcellsreinforcetheconclusionthatthearchaeaaremorecloselyrelatedtoeukaryoticcellsthantotheprokaryoticbacteria.,HighFidelityofReplication,BalancedlevelsofdNTPs.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),