Gene-X-ppt(Gene10-基因十)--Chapter14

Chapter14DNAReplication14.1Introduction•topoisomerase–AnenzymethatchangesthenumberoftimesthetwostrandsinaclosedDNAmoleculecrosseachother.–ItdoesthisbycuttingtheDNA,passingDNAthroughthebreak,andresealingtheDNA.•replisome–ThemultiproteinstructurethatassemblesatthebacterialreplicationforktoundertakesynthesisofDNA.–ItcontainsDNApolymeraseandotherenzymes.14.1IntroductionFIGURE01:Thereplisomeassemblesattheorigin14.1Introduction•conditionallethal–Amutationthatislethalunderonesetofconditions,butisnotlethalunderasecondsetofconditions,suchastemperature.•dnamutants-Temperature-sensitivereplicationmutantsinE.colithatidentifyasetoflocicalledthednagenes.•quick-stopmutant–Temperature-sensitivereplicationmutantsthataredefectiveinreplicationelongationduringsynthesisofDNA.14.1Introduction•slow-stopmutant–Temperature-sensitivereplicationmutantsthataredefectiveininitiationofreplication.•invitrocomplementation–Afunctionalassayusedtoidentifycomponentsofaprocess.–Thereactionisreconstructedusingextractsfromamutantcell.–Fractionsfromwild-typecellsarethentestedforrestorationofactivity.14.2Initiation:CreatingtheReplicationForksattheOriginoriC•licensingfactor–Afactorlocatedinthenucleusandnecessaryforreplicationthatisinactivatedordestroyedafteroneroundofreplication.–Newfactorsmustbeprovidedforfurtherroundsofreplicationtooccur.14.2Initiation:CreatingtheReplicationForksattheOriginoriC•InitiationatoriCrequiresthesequentialassemblyofalargeproteincomplexonthemembrane.•oriCmustbefullymethylated.•DnaA-ATPbindstoshortrepeatedsequencesandformsanoligomericcomplexthatmeltsDNA.FIGURE02:Theminimaloriginisdefinedbythedistancebetweentheoutsidemembersofthe13-merand9-merrepeats14.2Initiation:CreatingtheReplicationForksattheOriginoriC•SixDnaCmonomersbindeachhexamerofDnaB,andthiscomplexbindstotheorigin.FIGURE03:Prepriminginvolvesformationofacomplexbysequentialassociationofproteins,whichleadstotheseparationofDNAstrands14.2Initiation:CreatingtheReplicationForksattheOriginoriC•AhexamerofDnaBformsthereplicationfork.GyraseandSSBarealsorequired.•AshortregionofA-T-richDNAismelted.•DnaGisboundtothehelicasecomplexandcreatesthereplicationforks.•primase–AtypeofRNApolymerasethatsynthesizesshortsegmentsofRNAthatwillbeusedasprimersforDNAreplication.14.3DNAPolymerasesAretheEnzymesThatMakeDNA•DNAissynthesizedinbothsemiconservativereplicationandDNArepairreactions.FIGURE04:EachparentalstrandofDNAisatemplate14.3DNAPolymerasesAretheEnzymesThatMakeDNAFIGURE05:RepairsynthesisreplacesashortstretchofonestrandofDNAcontainingadamagedbase14.3DNAPolymerasesAretheEnzymesThatMakeDNAFIGURE06:DNAissynthesizedbyaddingnucleotidestothe3’–OHendofthegrowingchain,sothatthenewchaingrowsinthe5’to3’direction14.3DNAPolymerasesAretheEnzymesThatMakeDNA•AbacteriumoreukaryoticcellhasseveraldifferentDNApolymerase(DNAreplicase)enzymes.•OnebacterialDNApolymeraseundertakessemiconservativereplication;theothersareinvolvedinrepairreactions.FIGURE07:OnlyoneDNApolymeraseisthereplicationenzyme14.4DNAPolymerasesHaveVariousNucleaseActivities•DNApolymeraseIhasaunique5′–3′exonucleaseactivitythatcanbecombinedwithDNAsynthesistoperformnicktranslation.FIGURE08:NicktranslationreplacespartofapreexistingstrandofduplexDNAwithnewlysynthesizedmaterial14.5DNAPolymerasesControltheFidelityofReplication•High-fidelityDNApolymerasesinvolvedinreplicationhaveapreciselyconstrainedactivesitethatfavorsbindingofWatson–Crickbasepairs.•processivity–Theabilityofanenzymetoperformmultiplecatalyticcycleswithasingletemplateinsteadofdissociatingaftereachcycle.14.5DNAPolymerasesControltheFidelityofReplication•DNApolymerasesoftenhavea3′–5′exonucleaseactivitythatisusedtoexciseincorrectlypairedbases.•Thefidelityofreplicationisimprovedbyproofreadingbyafactorof~100.FIGURE09:BacterialDNApolymerasesscrutinizethebasepairattheendofthegrowingchainandexcisethenucleotideaddedinthecaseofamisfit14.6DNAPolymerasesHaveaCommonStructure•ManyDNApolymeraseshavealargecleftcomposedofthreedomainsthatresembleahand.•DNAliesacrossthe“palm”inagroovecreatedbythe“fingers”and“thumb.”FIGURE10:ThestructureoftheKlenowfragmentfromE.coliDNApolymeraseIStructurefromProteinDataBank1KFD.L.S.Beese,J.M.Friedman,andT.A.Steitz,Biochemistry32(1993):14095-14101.14.7TheTwoNewDNAStrandsHaveDifferentModesofSynthesis•TheDNApolymeraseadvancescontinuouslywhenitsynthesizestheleadingstrand(5′–3′),butsynthesizesthelaggingstrandbymakingshortfragments(Okasakifragments)thataresubsequentlyjoinedtogether.•semidiscontinuousreplication–Themodeofreplicationinwhichonenewstrandissynthesizedcontinuouslywhiletheotherissynthesizeddiscontinuously.14.7TheTwoNewDNAStrandsHaveDifferentModesofSynthesisFIGURE12:Theleadingstrandissynthesizedcontinuously,whereasthelaggingstrandissynthesizeddiscontinuously14.8ReplicationRequiresaHelicaseandaSingle-StrandBindingProtein•ReplicationrequiresahelicasetoseparatethestrandsofDNAusingenergyprovidedbyhydrolysisofATP.•Asingle-strandedbindingproteinisrequiredtomaintaintheseparatedstrands.FIGURE13:AhexamerichelicasemovesalongonestrandofDNA14.9PrimingIsRequiredtoStartDNASynthesis•A