武汉大学09年《基因的分子生物学》讲义chapter8.ppt

WelcomeEachofYoutoMyMolecularBiologyClass MolecularBiologyoftheGene 5 E Watsonetal 2004 PartI ChemistryandGeneticsPartII MaintenanceoftheGenomePartIII ExpressionoftheGenomePartIV RegulationPartV Methods PartII MaintenanceoftheGenome DedicatedtothestructureofDNAandtheprocessesthatpropagate maintainandalteritfromonecellgenerationtothenext Ch6 ThestructuresofDNAandRNACh7 Chromosomes chromatinsandthenucleosomeCh8 ThereplicationofDNACh9 ThemutabilityandrepairofDNACh10 HomologousrecombinationatthemolecularlevelCh11 Site specificrecombinationandtranspositionofDNA CHAPTER8 ThereplicationofDNA MolecularBiologyCourse HowDNAmoleculessustainingthebeautifullivingcreaturesarepropagatedfromonecellgenerationtothenext Appreciate Thesimplebeautyofthereplicationchemistry ThefineartofDNApolymeraseTheharmoniouscoordinationbetweenmanyproteinfactors Thestrictcontroloftheinitiationandthecompletenessoftermination TeachingArrangement Watchanimation UnderstandreplicationGothroughsomestructuraltutorial ExperiencetheBEAUTYoftheDNApolymeraseLecture comprehensiveunderstandingandhighlightKeypoints CHAPTER8ThereplicationofDNA TheChemistryofDNASynthesisTheMechanismofDNAPolymeraseTheSpecializationofDNAPolymerasesTheReplicationForkDNASynthesisattheReplicationForkInitiationofDNAReplicationBindingandUnwindingFinishingReplication CHAPTER8ThereplicationofDNA I Reaction Catalyst II Replicationatafork III Initiation Termination ThefirstpartdescribesthebasicchemistryofDNAsynthesisandthefunctionoftheDNApolymerase CHAPTER8ThereplicationofDNA TheChemistryofDNA CHAPTER8ThereplicationofDNA DNAsynthesisrequiresdeoxynucleosidetriphosphatesandaprimer templatejunctionDNAissynthesizedbyextendingthe3 endoftheprimerHydrolysisofpyrophosphate PPi isthedrivingforceforDNAsynthesis Figure8 3SubstratesrequiredforDNAsynthesis ThemechanismofDNAPolymerase Pol CHAPTER8ThereplicationofDNA DNAPoluseasingleactivesitetocatalyzeDNAsynthesis AsinglesitetocatalyzetheadditionofanyofthefourdNTPs RecognitionofdifferentdNTPbymonitoringtheabilityofincomingdNTPinformingA TandG Cbasepairs incorrectbasepairdramaticallylowerstherateofcatalysis kineticselectivity ThemechanismofDNAPol Figure8 3 DistinguishingdifferentdNTPs kineticselectivity DistinguishingbetweenrNTPanddNTPbystericexclusionofrNTPsfromtheactivesite ThemechanismofDNAPol Figure8 4 DNAPolresembleahandthatgripstheprimer templatejunction ThemechanismofDNAPol Figure8 5 SchematicofDNApolboundtoaprimer templatejunction AsimilarviewoftheT7DNApolboundtoDNA Figure8 8 Thumb Fingers Palm Containstwocatalyticsites oneforadditionofdNTPsandoneforremovalofthemispaireddNTP Thepolymerizationsite 1 bindstotwometalionsthatalterthechemicalenvironmentaroundthecatalyticsiteandleadtothecatalysis how Figures8 6 8 7 2 Monitorstheaccuracyofbase pairingforthemostrecentlyaddednucleotidesbyformingextensivehydrogenbondcontactswithminorgrooveofthenewlysynthesizedDNA Exonucleasesite proofreadingsite Seeproofreading DNAPolymerase palmdomain Figure8 6 Figure8 7 BindstotheincomingdNTP enclosesthecorrectpaireddNTPtothepositionforcatalysisBendsthetemplatetoexposetheonlynucleotideatthetemplatethatreadyforformingbasepairwiththeincomingnucleotideStabilizationofthepyrophosphate DNAPolymerase fingerdomain NotdirectlyinvolvedincatalysisInteractswiththesynthesizedDNAtomaintaincorrectpositionoftheprimerandtheactivesite andtomaintainastrongassociationbetweenDNAPolanditssubstrate DNAPolymerase thumbdomain DNAPolareprocessiveenzymes ThemechanismofDNAPol Processivityisacharacteristicofenzymesthatoperateonpolymericsubstrates TheprocessivityofDNAPolistheaveragenumberofnucleotidesaddedeachtimetheenzymebindsaprimer templatejunction varyingfromafewto 50 000nucleotides TherateofDNAsynthesisiscloselyrelatedtothepolymeraseprocessivity becausetherate limitingstepistheinitialbindingofpolymerasetotheprimer templatejunction Figure8 9 ExonucleasesproofreadnewlysynthesizedDNA ThemechanismofDNAPol Theoccasionalflickingofthebasesinto wrong tautomericformresultsinincorrectbasepairandmis incorporationofdNTP 10 5mistake ThemismatcheddNMPisremovedbyproofreadingexonuclease apartoftheDNApolymerase Howdoestheexonucleaseswork Kineticselectivity Figure8 10 ThespecializationofDNApolymerases CHAPTER8ThereplicationofDNA DNAPolsarespecializedfordifferentrolesinthecell ThespecializationofDNApol EachorganismhasadistinctsetofdifferentDNAPolsDifferentorganismshavedifferentDNAPolsDNAPolIIIholoenzyme aproteincomplexresponsibleforE coligenomereplicationDNAPolI removesRNAprimersinE coli EukaryoticcellshavemultipleDNApolymerases Threeareessentialtoduplicatethegenome DNAPold DNAPoleandDNAPola primase Whataretheirfunctions PolymeraseswitchinginEukaryotes theprocessofreplacingDNAPola primasewithDNAPoldorDNAPole Table8 2 SlidingclampsdramaticallyincreaseDNApolymeraseactivity ThespecializationofDNApol Encirclethenewlysynthesizeddouble strandedDNAandthepolymeraseassociatedwiththeprimer templatejunctionEnsurestherapidrebindingofDNAPoltothesameprimer templatejunction andthusincreasestheprocessivityofPol p221fordetails EukaryoticslidingDNAclampisPCNA Figure8 17 Figure8 19SlidingDNAclampsarefoundacrossallorganismandshareasimilarstructure SlidingclampsareopenedandplacedonDNAbyclamploaders ThespecializationofDNApol ClamploaderisaspecialclassofproteincomplexcatalyzestheopeningandplacementofslidingclampsontheDNA suchaprocessoccursanytimeaprimer templatejunctionispresent SlidingclampsareonlyremovedfromtheDNAoncealltheassociatedenzymescompletetheirfunction Box8 4ATPcontrolofProteinFunction LoadingaSlidingClamp ThesecondpartdescribeshowthesynthesisofDNAoccursinthecontextofanintactchromosomeatreplicationforks AnarrayofproteinsarerequiredtoprepareDNAreplicationatthesesites CHAPTER8ThereplicationofDNA Thereplicationfork CHAPTER8ThereplicationofDNA ThejunctionbetweenthenewlyseparatedtemplatestrandsandtheunreplicatedduplexDNA BothstrandsofDNAaresynthesizedtogetheratthereplicationfork Thereplicationfork Figure8 11 Leadingstrand Laggingstrand Okazakifragment Replicationfork Thereplicationfork ReplicationforkenzymesextendtherangeofDNApolymerasesubstrate DNAPolcannotaccomplishreplicationwithoutthehelpofotherenzymesThebornanddeathofaRNAprimer primaseandRNaseH exonuclease DNAPol ligaseDealingtheDNAstructure helicase topoisomerase SSB TheinitiationofanewstrandofDNArequireanRNAprimer Thereplicationfork PrimaseisaspecializedRNApolymerasededicatedtomakingshortRNAprimersonanssDNAtemplate DonotrequirespecificDNAsequence DNAPolcanextendbothRNAandDNAprimersannealedtoDNAtemplate RNAprimersmustberemovedtocompleteDNAreplication Thereplicationfork AjointeffortsofRNaseH DNApolymerase DNAligase Figure8 12 Thereplicationfork Figure8 15 TopoisomeraseremovessupercoilsproducedbyDNAunwindingatthereplicationfork DNAhelicasesunwindthedoublehelixinadvanceofthereplicationfork Thereplicationfork Figure8 13 Single strandedbindingproteins SSBs stabilizesingle strandedDNA Thereplicationfork CooperativebindingSequence independentmanner electrostaticinteractions Figure8 14 DNAsynthesisatthereplicationfork CHAPTER8ThereplicationofDNA Theleadingstrandandlaggingstrandaresynthesizedsimultaneously Atthereplication theleadingstrandandlaggingstrandaresynthesizedsimultaneously ThebiologicalrelevanceislistedinP205 206Tocoordinatethereplicationofbothstrands multipleDNAPolsfunctionatthereplicationfork DNAPolIIIholoenzymeissuchanexample Figure8 20ThecompositionoftheDNAPolIIIholoenzyme Figure8 21 Trombonemodel InteractionsbetweenreplicationforkproteinsformtheE colireplisome DNAsynthesisatthereplicationfork Replisomeisestablishedbyprotein proteininteractionsDNAhelicase DNAPolIIIholoenzyme thisinteractionismediatedbytheclamploaderandstimulatestheactivityofthehelicase 10 fold DNAhelicase primase whichisrelativelyweekandstronglystimulatestheprimasefunction 1000 fold ThisinteractionisimportantforregulationthelengthofOkazakifragments DNAPolIIIholoenzyme helicaseandprimaseinteractwitheachothertoformreplisome afinelytunedfactoryforDNAsynthesiswiththeactivityofeachproteinishighlycoordinated ThethirdpartfocusesontheinitiationandterminationofDNAreplication DNAreplicationistightlycontrolledinallcellsandinitiationisthestepforregulation CHAPTER8ThereplicationofDNA CHAPTER8ThereplicationofDNA InitiationofDNAreplication CHAPTER8ThereplicationofDNA SpecificgenomicDNAsequencesdirecttheinitiationofDNAreplication Originsofreplication thesitesatwhichDNAunwindingandinitiationofreplicationoccur InitiationofDNAreplication Therepliconmodelofreplicationinitiation ageneralview ProposedbyJacobandBrennerin1963AlltheDNAreplicatedfromaparticularoriginisarepliconTwocomponents replicatorandinitiator controltheinitiationofreplication InitiationofDNAreplication Replicator theentiresiteofcis actingDNAsequencessufficienttodirecttheinitiationofDNAreplication Initiatorprotein specificallyrecognizesaDNAelementinthereplicatorandactivatestheinitiationofreplication Figure8 23 ReplicatorsequencesincludeinitiatorbindingsitesandeasilyunwoundDNA BindingandUnwinding originselectionandactivationbytheinitiatorprotein CHAPTER8ThereplicationofDNA Threedifferentfunctionsofinitiatorprotein 1 bindstoreplicator 2 distorts unwindsaregionofDNA 3 interactswithandrecruitsadditionalreplicationfactorsDnaAinE coli all3functions originrecognitioncomplex ORC ineukaryotes functions1 3 Protein proteinandprotein DNAinteractionsdirecttheinitiationprocess Bindingandunwinding Initiatingreplicationinbacteria DnaArecruitstheDNAhelicaseDnaBandthehelicaseloaderDnaCDnaBinteractswithprimasetoinitiateRNAprimersynthesis Figure8 27 InitiatingreplicationineukaryotesEukaryoticchromosomearereplicatedexactlyoncepercellcycle whichiscriticalfortheseorganisms Bindingandunwinding Pre replicativecomplex pre RC formationandactivationdirectstheinitiationofreplicationineukaryotes Initiationineukaryotesrequirestwodistinctsteps 1ststep Replicatorselection theprocessofidentifyingsequencesforreplicationinitiation G1phase whichismediatedbytheformationofpre RCsatthereplicatorregion Figure8 30pre RCformation 2ndstep Originactivation pre RCsareactivatedbytwoproteinkinases CdkandDdk thatareactiveonlywhenthecellsenterSphase Figure8 31 Activationofthepre RCleadstotheassemblyoftheeukaryoticreplicationfork Pre RCformationandactivationistightlyregulatedtoallowonlyasingleroundofreplicationduringeachcellcycle Onlyoneopportunityforpre RCstoform andonlyoneopportunityforpre RCactivation Figure8 32EffectofCdkactivityonpre RCformationandactivation Figure8 33CellcycleregulationofCdkactivityandpre RCformatin Finishingreplication CHAPTER8ThereplicationofDNA Finishingreplicationinbacteria TypeIItopoisomerasesseparatedaughterDNAmolecules Finishingreplication Figure8 34TopoisomeraseIIcatalyzethedecatenationofreplicationproducts Finishingreplicationineukaryotes TheendreplicationproblemTelomere telomerase alinkwithcancerandaging Finishingreplication Whatistheendreplicationproblem Laggingstrandsynthesisisunabletocopytheextremeendsofthelinearchromosome Figure8 34 TelomeraseisanovelDNApolymerasethatdoesnotrequireanexogenoustemplate Howtelomeraseworks Telomeraseextendstheprotruding3 endofthechromosomeusingitsRNAcomponentsasatemplate Figure8 37 Howtheendproblemiseventuallyresolved Figure8 38 Theextended3 endallowstheDNApolymerasetosynthesizeanewOkazakifragment whichpreventsthelossofgeneticinformationatthechromosomalend Figure8 39 Telomere bindingproteins Telomere bindingproteinsregulatetelomeraseactivityandtelomerelength Figure8 40 Telomerelengthregulationbytelomere bindingproteins Shorttelomereisboundbyfewtelomere bindingproteins allowingthetelomerasetoextendtelomere Theextendedtelomereisboundbymoretelomere bindingproteins whichinhibitsthetelomeraseactivity 重点 CHAPTER8ThereplicationofDNA Completelyunderstand三个AnimationsDNApolymerization Topics1 2 DNAreplication Topics3 5 ActionofTelomerase Topic8 TheChemistryofDNASynthesis substrate directionandenergy TheMechanismofDNAPolymerase 1polymerizationmechanism 2differentwaysofdiscriminatingsubstrates 2catalyticsites 3domains TheSpecializationofDNAPolymerasesTheReplicationFork theenzyme proteinsrequiredtosynthesizetheleadingandlaggingstrands DNASynthesisattheR