DNA-replication

WelcomeEachofYoutoMyMolecularBiologyClassMolecularBiologyoftheGene,5/E---Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenomePartIII:ExpressionoftheGenomePartIV:RegulationPartV:MethodsPartII:MaintenanceoftheGenomeDedicatedtothestructureofDNAandtheprocessesthatpropagate,maintainandalteritfromonecellgenerationtothenextCh6:ThestructuresofDNAandRNACh7:Chromosomes,chromatinsandthenucleosomeCh8:ThereplicationofDNACh9:ThemutabilityandrepairofDNACh10:HomologousrecombinationatthemolecularlevelCh11:Site-specificrecombinationandtranspositionofDNACHAPTER8:ThereplicationofDNA•MolecularBiologyCourseHowDNAmoleculessustainingthebeautifullivingcreaturesarepropagatedfromonecellgenerationtothenext…TeachingArrangementWatchanimation-UnderstandreplicationGothroughsomestructuraltutorial-ExperiencetheBEAUTYoftheDNApolymeraseLecture-comprehensiveunderstandingandhighlightKeypointsCHAPTER8ThereplicationofDNA1.TheChemistryofDNASynthesis2.TheMechanismofDNAPolymerase3.TheSpecializationofDNAPolymerases4.TheReplicationFork5.DNASynthesisattheReplicationFork7.InitiationofDNAReplication8.BindingandUnwinding9.FinishingReplicationCHAPTER8ThereplicationofDNAI.Reaction&CatalystII.ReplicationataforkIII.Initiation&TerminationThefirstpartdescribesthebasicchemistryofDNAsynthesisandthefunctionoftheDNApolymeraseCHAPTER8ThereplicationofDNATheChemistryofDNACHAPTER8ThereplicationofDNADNAsynthesisrequiresdeoxynucleosidetriphosphatesandaprimer:templatejunctionDNAissynthesizedbyextendingthe3’endoftheprimerHydrolysisofpyrophosphate(PPi)isthedrivingforceforDNAsynthesisFigure8-3SubstratesrequiredforDNAsynthesisThemechanismofDNAPolymerase(Pol)CHAPTER8ThereplicationofDNADNAPoluseasingleactivesitetocatalyzeDNAsynthesisAsinglesitetocatalyzetheadditionofanyofthefourdNTPs.RecognitionofdifferentdNTPbymonitoringtheabilityofincomingdNTPinformingA-TandG-Cbasepairs;incorrectbasepairdramaticallylowerstherateofcatalysis(kineticselectivity).ThemechanismofDNAPolFigure8-3DistinguishingdifferentdNTPs:kineticselectivityDistinguishingbetweenrNTPanddNTPbystericexclusionofrNTPsfromtheactivesite.ThemechanismofDNAPolFigure8-4DNAPolresembleahandthatgripstheprimer-templatejunctionThemechanismofDNAPolFigure8-5SchematicofDNApolboundtoaprimer:templatejunctionAsimilarviewoftheT7DNApolboundtoDNAFigure8-8ThumbFingersPalm1.Containstwocatalyticsites,oneforadditionofdNTPsandoneforremovalofthemispaireddNTP.2.Thepolymerizationsite:(1)bindstotwometalionsthatalterthechemicalenvironmentaroundthecatalyticsiteandleadtothecatalysis.(how?Figures8-6,8-7).(2)Monitorstheaccuracyofbase-pairingforthemostrecentlyaddednucleotidesbyformingextensivehydrogenbondcontactswithminorgrooveofthenewlysynthesizedDNA.3.Exonucleasesite/proofreadingsite(Seeproofreading)DNAPolymerase-palmdomainFigure8-6Figure8-7BindstotheincomingdNTP,enclosesthecorrectpaireddNTPtothepositionforcatalysisBendsthetemplatetoexposetheonlynucleotideatthetemplatethatreadyforformingbasepairwiththeincomingnucleotideStabilizationofthepyrophosphateDNAPolymerase-fingerdomainNotdirectlyinvolvedincatalysisInteractswiththesynthesizedDNAtomaintaincorrectpositionoftheprimerandtheactivesite,andtomaintainastrongassociationbetweenDNAPolanditssubstrate.DNAPolymerase-thumbdomainDNAPolareprocessiveenzymesThemechanismofDNAPolProcessivityisacharacteristicofenzymesthatoperateonpolymericsubstrates.TheprocessivityofDNAPolistheaveragenumberofnucleotidesaddedeachtimetheenzymebindsaprimer:templatejunction(varyingfromafewto50,000nucleotides).TherateofDNAsynthesisiscloselyrelatedtothepolymeraseprocessivity,becausetherate-limitingstepistheinitialbindingofpolymerasetotheprimer-templatejunction.Figure8-9ExonucleasesproofreadnewlysynthesizedDNAThemechanismofDNAPolTheoccasionalflickingofthebasesinto“wrong”tautomericformresultsinincorrectbasepairandmis-incorporationofdNTP.(10-5mistake)ThemismatcheddNMPisremovedbyproofreadingexonuclease,apartoftheDNApolymerase.Howdoestheexonucleaseswork?KineticselectivityFigure8-10ThespecializationofDNApolymerasesCHAPTER8ThereplicationofDNADNAPolsarespecializedfordifferentrolesinthecellThespecializationofDNApolEachorganismhasadistinctsetofdifferentDNAPolsDifferentorganismshavedifferentDNAPolsDNAPolIIIholoenzyme:aproteincomplexresponsibleforE.coligenomereplicationDNAPolI:removesRNAprimersinE.coliEukaryoticcellshavemultipleDNApolymerases.Threeareessentialtoduplicatethegenome:DNAPold,DNAPoleandDNAPola/primase.(Whataretheirfunctions?)PolymeraseswitchinginEukaryotes:theprocessofreplacingDNAPola/primasewithDNAPoldorDNAPole.Table8-2***SlidingclampsdramaticallyincreaseDNApolymeraseactivityThespecializationofDNApolEncirclethenewlysynthesizeddouble-strandedDNAandthepolymeraseassociatedwiththeprimer:templatejunctionEnsurestherapidrebindingofDNAPoltothesameprimer:templatejunction,andthusincreasestheprocessivityofPol.[p221fordetails]EukaryoticslidingDNAclampisPCNAFigure8-17Figure8-19SlidingDNAclampsarefoundacrossallorganismandshareasimilarstructureSlidingclampsareopenedandplacedonDNAbyclamploadersThespecializationofDNApolCla