WelcomeEachofYoutoMyMolecularBiologyClassMolecularBiologyoftheGene,5/E

---Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenome

PartIII:ExpressionoftheGenomePartIV:RegulationPartV:MethodsPartII:MaintenanceoftheGenomeDedicatedtothestructureofDNAandtheprocessesthatpropagate,maintainandalteritfromonecellgenerationtothenextCh6:ThestructuresofDNAandRNACh7:Chromosomes,chromatinsandthenucleosomeCh8:ThereplicationofDNACh9:ThemutabilityandrepairofDNACh10:HomologousrecombinationatthemolecularlevelCh11:Site-specificrecombinationandtranspositionofDNACHAPTER8:ThereplicationofDNAMolecularBiologyCourseTeachingArrangementWatchanimation-UnderstandreplicationGothroughsomestructuraltutorial-ExperiencetheBEAUTYoftheDNApolymeraseLecture-comprehensiveunderstanding

andhighlightKeypointsCHAPTER8ThereplicationofDNATheChemistryofDNASynthesisTheMechanismofDNAPolymeraseTheSpecializationofDNAPolymerasesTheReplicationForkDNASynthesisattheReplicationForkInitiationofDNAReplicationBindingandUnwindingFinishingReplicationProcessInitiation&TerminationCHAPTER8ThereplicationofDNAReaction&CatalystThefirstpartdescribesthebasicchemistryofDNAsynthesisandthefunctionoftheDNApolymeraseCHAPTER8ThereplicationofDNATheChemistryofDNACHAPTER8ThereplicationofDNADNAsynthesisrequiresdeoxynucleosidetriphosphatesandaprimer:templatejunctionDNAissynthesizedbyextendingthe3’endoftheprimerHydrolysisofpyrophosphate(PPi)isthedrivingforceforDNAsynthesisFigure8-3SubstratesrequiredforDNAsynthesisThemechanismofDNAPolymerase(Pol)CHAPTER8ThereplicationofDNADNAPoluseasingleactivesitetocatalyzeDNAsynthesisAsinglesitetocatalyzetheadditionofanyofthefourdNTPs.RecognitionofdifferentdNTPbymonitoringtheabilityofincomingdNTPinformingA-TandG-Cbasepairs;incorrectbasepairdramaticallylowerstherateofcatalysis(kineticselectivity).ThemechanismofDNAPolFigure8-3DistinguishingdifferentdNTPs:kineticselectivityDistinguishingbetweenrNTPanddNTPbystericexclusionofrNTPsfromtheactivesite.ThemechanismofDNAPolFigure8-4DNAPolresembleahandthatgripstheprimer-templatejunctionThemechanismofDNAPolFigure8-5SchematicofDNApolboundtoaprimer:templatejunctionAsimilarviewoftheT7DNApolboundtoDNAFigure8-8ThumbFingersPalmContainstwocatalyticsites,oneforadditionofdNTPsandoneforremovalofthemispaireddNTP.Thepolymerizationsite:(1)

bindstotwometalionsthatalterthechemicalenvironmentaroundthecatalyticsiteandleadtothecatalysis.(how?Figures8-6,8-7).(2)Monitorstheaccuracyofbase-pairingforthemostrecentlyaddednucleotidesbyformingextensivehydrogenbondcontactswithminorgrooveofthenewlysynthesizedDNA.Exonucleasesite/proofreadingsite(Seeproofreading)DNAPolymerase-palmdomainFigure8-6Figure8-7BindstotheincomingdNTP,enclosesthecorrectpaireddNTPtothepositionforcatalysisBendsthetemplatetoexposetheonlynucleotideatthetemplatethatreadyforformingbasepairwiththeincomingnucleotideStabilizationofthepyrophosphateDNAPolymerase-fingerdomainNotdirectlyinvolvedincatalysisInteractswiththesynthesizedDNAtomaintaincorrectpositionoftheprimerandtheactivesite,andtomaintainastrongassociationbetweenDNAPolanditssubstrate.DNAPolymerase-thumbdomainDNAPolareprocessiveenzymesThemechanismofDNAPolProcessivityisacharacteristicofenzymesthatoperateonpolymericsubstrates.TheprocessivityofDNAPolistheaveragenumberofnucleotidesaddedeachtimetheenzymebindsaprimer:templatejunction(varyingfromafewto>50,000nucleotides).TherateofDNAsynthesisiscloselyrelatedtothepolymeraseprocessivity,becausetherate-limitingstepistheinitialbindingofpolymerasetotheprimer-templatejunction.Figure8-9ExonucleasesproofreadnewlysynthesizedDNAThemechanismofDNAPolTheoccasionalflickingofthebasesinto“wrong”tautomericformresultsinincorrectbasepairandmis-incorporationofdNTP.(10-5mistake)ThemismatcheddNMPisremovedbyproofreadingexonuclease,apartoftheDNApolymerase.Howdoestheexonucleaseswork?KineticselectivityFigure8-10ThespecializationofDNApolymerasesCHAPTER8ThereplicationofDNADNAPolsarespecializedfordifferentrolesinthecellThespecializationofDNApolEachorganismhasadistinctsetofdifferentDNAPolsDifferentorganismshavedifferentDNAPolsDNAPolIIIholoenzyme:aproteincomplexresponsibleforE.coligenomereplicationDNAPolI:removesRNAprimersinE.coli

EukaryoticcellshavemultipleDNApolymerases.Threeareessentialtoduplicatethegenome:DNAPold,DNAPoleandDNAPola/primase.(Whataretheirfunctions?)PolymeraseswitchinginEukaryotes:theprocessofreplacingDNAPola/primasewithDNAPoldorDNAPole.Table8-2***SlidingclampsdramaticallyincreaseDNApolymeraseactivityThespecializationofDNApolEncirclethenewlysynthesizeddouble-strandedDNAandthepolymeraseassociatedwiththeprimer:templatejunctionEnsurestherapidrebindingofDNAPoltothesameprimer:templatejunction,andthusincreasestheprocessivityofPol.[p221fordetails]EukaryoticslidingDNAclampisPCNAFigure8-17Figure8-19SlidingDNAclampsarefoundacrossallorganismandshareasimilarstructureSlidingclampsareopenedandplacedonDNAbyclamploadersThespecializationofDNApolClamploaderisaspecialclassofproteincomplexcatalyzestheopeningandplacementofslidingclampsontheDNA,suchaprocessoccursanytimeaprimer:templatejunctionispresent.SlidingclampsareonlyremovedfromtheDNAoncealltheassociatedenzymescompletetheirfunction.Box8-4ATPcontrolofProteinFunction:LoadingaSlidingClampThesecondpartdescribeshowthesynthesisofDNAoccursinthecontextofanintactchromosomeatreplicationforks.AnarrayofproteinsarerequiredtoprepareDNAreplicationatthesesites.CHAPTER8ThereplicationofDNAThereplicationforkCHAPTER8ThereplicationofDNAThejunctionbetweenthenewlyseparatedtemplatestrandsandtheunreplicatedduplexDNABothstrandsofDNAaresynthesizedtogetheratthereplicationfork.ThereplicationforkFigure8-11LeadingstrandLaggingstrandOkazakifragmentReplicationforkThereplicationforkReplicationforkenzymesextendtherangeofDNApolymerasesubstrateDNAPolcannotaccomplishreplicationwithoutthehelpofotherenzymesThebornanddeathofaRNAprimer:primaseandRNaseH/exonuclease/DNAPol/ligaseDealingtheDNAstructure(helicase,topoisomerase,SSB)TheinitiationofanewstrandofDNArequireanRNAprimerThereplicationforkPrimaseisaspecializedRNApolymerasededicatedtomakingshortRNAprimersonanssDNAtemplate.DonotrequirespecificDNAsequence.DNAPolcanextendbothRNAandDNAprimersannealedtoDNAtemplateRNAprimersmustberemovedtocompleteDNAreplicationThereplicationforkAjointeffortsofRNaseH,DNApolymerase&DNAligaseFigure8-12ThereplicationforkFigure8-15TopoisomeraseremovessupercoilsproducedbyDNAunwindingatthereplicationforkDNAhelicasesunwindthedoublehelixinadvanceofthereplicationforkThereplicationforkFigure8-13Single-strandedbindingproteins(SSBs)stabilizesingle-strandedDNAThereplicationforkCooperative

bindingSequence-independentmanner(electrostaticinteractions)Figure8-14DNAsynthesisatthereplicationforkCHAPTER8ThereplicationofDNATheleadingstrandandlaggingstrandaresynthesizedsimultaneously.Atthereplication,theleadingstrandandlaggingstrandaresynthesizedsimultaneously.ThebiologicalrelevanceislistedinP205-206Tocoordinatethereplicationofbothstrands,multipleDNAPolsfunctionatthereplicationfork.DNAPolIIIholoenzymeissuchanexample.Figure8-20ThecompositionoftheDNAPolIIIholoenzymeFigure8-21***TrombonemodelInteractionsbetweenreplicationforkproteinsformtheE.coli

replisomeDNAsynthesisatthereplicationforkReplisomeisestablishedbyprotein-proteininteractionsDNAhelicase&DNAPolIIIholoenzyme,thisinteractionismediatedbytheclamploaderandstimulatestheactivityofthehelicase(10-fold)DNAhelicase&primase,whichisrelativelyweekandstronglystimulatestheprimasefunction(1000-fold).ThisinteractionisimportantforregulationthelengthofOkazakifragments.DNAPolIIIholoenzyme,helicaseandprimaseinteractwitheachothertoformreplisome,afinelytunedfactoryforDNAsynthesiswiththeactivityofeachproteinishighlycoordinated.ThethirdpartfocusesontheinitiationandterminationofDNAreplication.NotethatDNAreplicationistightlycontrolledinallcellsandinitiationisthestepforregulation.CHAPTER8ThereplicationofDNAInitiationofDNAreplicationCHAPTER8ThereplicationofDNASpecificgenomicDNAsequencesdirecttheinitiationofDNAreplicationOriginsofreplication,thesitesatwhichDNAunwindingandinitiationofreplicationoccur.InitiationofDNAreplicationTherepliconmodelofreplicationinitiation---ageneralviewProposedbyJacobandBrennerin1963AlltheDNAreplicatedfromaparticularoriginisarepliconTwocomponents,replicatorandinitiator,controltheinitiationofreplicationInitiationofDNAreplicationReplicator:theentiresiteofcis-actingDNAsequencessufficienttodirecttheinitiationofDNAreplicationInitiatorprotein:specificallyrecognizesaDNAelementinthereplicatorandactivatestheinitiationofreplicationFigure8-23ReplicatorsequencesincludeinitiatorbindingsitesandeasilyunwoundDNABindingandUnwinding:originselectionandactivationbytheinitiatorproteinCHAPTER8ThereplicationofDNAThreedifferentfunctionsofinitiatorprotein:(1)bindstoreplicator,(2)distorts/unwindsaregionofDNA,(3)interactswithandrecruitsadditionalreplicationfactorsDnaAinE.coli(all3functions),originrecognitioncomplex(ORC)ineukaryotes(functions1&3)Protein-proteinandprotein-DNAinteractionsdirecttheinitiationprocessBindingandunwindingInitiatingreplicationinbacteriaDnaArecruitstheDNAhelicaseDnaBandthehelicaseloaderDnaCDnaBinteractswithprimasetoinitiateRNAprimersynthesis.Figure8-27*InitiatingreplicationineukaryotesEukaryoticchromosomearereplicatedexactlyoncepercellcycle,whichiscriticalfortheseorganismsBindingandunwindingPre-replicativecomplex(pre-RC)formationandactivationdirectstheinitiationofreplicationineukaryotesInitiationineukaryotesrequirestwodistinctsteps:1ststep---Replicatorselection:theprocessofidentifyingsequencesforreplicationinitiation(G1phase),whichismediatedbytheformationofpre-RCsatthereplicatorregion.Figure8-30pre-RCformation2ndstep---Originactivation:pre-RCsareactivatedbytwoproteinkinases(CdkandDdk)thatareactiveonlywhenthecellsenterSphase.Figure8-31:Activationofthepre-RCleadstotheassemblyoftheeukaryoticreplicationfork.Pre-RCformationandactivationistightlyregulatedtoallowonlyasingleroundofreplicationduringeachcellcycle.Onlyoneopportunityforpre-RCstoform,andonlyoneopportunityforpre-RCactivation.Figure8-32EffectofCdkactivityonpre-RCformationandactivationFigure8-33CellcycleregulationofCdkactivityandpre-RCformatinFinishingreplicationCHAPTER8ThereplicationofDNAFinishingreplicationinbacteria:TypeIItopoisomerasesseparatedaughterDNAmoleculesFinishingreplicationFigure8-34TopoisomeraseIIcatalyzethedecatenationofreplicationproducts.Finishingreplicationineukaryotes:TheendreplicationproblemTelomere&telomerase:alinkwithcancerandagingFinishingreplicationWhatistheendreplicationproblem?LaggingstrandsynthesisisunabletocopytheextremeendsofthelinearchromosomeFigure8-34TelomeraseisanovelDNApolymerasethatdoesnotrequireanexogenoustemplateHowtelomeraseworks?

Telomeraseextendstheprotruding3’endofthechromosomeusingitsRNAcomponentsasatemplate.

(Figure8-37)

Howtheendproblemiseventuallyresolved?Figure8-38Theextended3’endallowstheDNApolymerasetosynthesizeanewOkazakifragment,whichpreventsthelossofgeneticinformationatthechromosomalend.Figure8-39:Telomere-bindingproteins.Telomere-bindingproteinsregulatetelomeraseactivityandtelomerelengthFigure8-40:Telomerelengthregulationbytelomere-bindingproteins.Shorttelomereisboundbyfewtelomere-bindingproteins,allowingthetelomerasetoextendtelomere.Theextendedtelomereisboundbymoretelomere-bindingproteins,whichinhibitsthetelomeraseactivity.重點(diǎn)CHAPTER8ThereplicationofDNACompletelyunderstand三個(gè)AnimationsDNApolymerization(Topics1&2)DNAreplication(Topics3－5)ActionofTelomerase(Topic8)TheChemistryofDNASynthesis:substrate,directionandenergy.TheMechanismofDNAPolymerase:1polymerizationmechanism,2differentwaysofdiscriminatingsubstrates,2catalyticsites;3domains.TheSpecializationofDNAPolymerasesTheReplicationFork:theenzyme/proteinsrequiredtosynthesizetheleadingandlaggingstrands.DNASynthesisattheReplicationFork:Holoenzyme/trombonemodeltoexplainhowtheanti-paralleltemplatestrands