DNA Replication.ppt

Part3GeneticInformationTransferDNARNAproteintranscriptiontranslationreplicationreversetranscriptionCentraldogma•Replication:synthesisofdaughterDNAfromparentalDNA•Transcription:synthesisofRNAusingDNAasthetemplate•Translation:proteinsynthesisusingmRNAmoleculesasthetemplate•Reversetranscription:synthesisofDNAusingRNAasthetemplateChapter10DNAReplicationSection1GeneralConceptsofDNAReplicationDNAreplication•AreactioninwhichdaughterDNAsaresynthesizedusingtheparentalDNAsasthetemplate.•TransferringthegeneticinformationtothedescendantgenerationwithahighfidelityreplicationparentalDNAdaughterDNADaughterstrandsynthesis•Chemicalformulation:(dNMP)n+dNTP(dNMP)n+1+PPiDNAstrandsubstrateelongatedDNAstrand•ThenatureofDNAreplicationisaseriesof3´-5´phosphodiesterbondformationcatalyzedbyagroupofenzymes.PhosphodiesterbondformationTemplate:doublestrandedDNASubstrate:dNTPPrimer:shortRNAfragmentwithafree3´-OHendEnzyme:DNA-dependentDNApolymerase(DDDP),otherenzymes,proteinfactorDNAreplicationsystemCharacteristicsofreplicationSemi-conservativereplicationBidirectionalreplicationSemi-continuousreplicationHighfidelity§1.1Semi-ConservativeReplicationSemiconservativereplicationHalfoftheparentalDNAmoleculeisconservedineachnewdoublehelix,pairedwithanewlysynthesizedcomplementarystrand.ThisiscalledsemiconservativereplicationSemiconservativereplicationExperimentofDNAsemiconservativereplicationHeavyDNA(15N)growin14NmediumThefirstgenerationgrowin14NmediumThesecondgenerationSignificanceThegeneticinformationisensuredtobetransferredfromonegenerationtothenextgenerationwithahighfidelity.§1.2BidirectionalReplication•ReplicationstartsfromunwindingthedsDNAataparticularpoint(calledorigin),followedbythesynthesisoneachstrand.•TheparentaldsDNAandtwonewlyformeddsDNAformaY-shapestructurecalledreplicationfork.3'5'5'3'5'3'5'3'directionofreplicationReplicationforkBidirectionalreplication•OncethedsDNAisopenedattheorigin,tworeplicationforksareformedspontaneously.•Thesetworeplicationforksmoveinoppositedirectionsasthesynthesescontinue.BidirectionalreplicationReplicationofprokaryotesThereplicationprocessstartsfromtheorigin,andproceedsintwooppositedirections.Itisnamedreplication.Replicationofeukaryotes•Chromosomesofeukaryoteshavemultipleorigins.•Thespacebetweentwoadjacentoriginsiscalledthereplicon,afunctionalunitofreplication.5'3'3'5'bidirectionalreplicationfusionofbubbles3'5'3'5'5'3'5'3'originsofDNAreplication(every~150kb)§1.3Semi-continuousReplicationThedaughterstrandsontwotemplatestrandsaresynthesizeddifferentlysincethereplicationprocessobeystheprinciplethatDNAissynthesizedfromthe5´endtothe3´end.5'3'3'5'5'directionofunwinding3'Onthetemplatehavingthe3´-end,thedaughterstrandissynthesizedcontinuouslyinthe5’-3’direction.Thisstrandisreferredtoastheleadingstrand.LeadingstrandSemi-continuousreplication3'5'5'3'replicationdirectionOkazakifragment3'5'leadingstrand3'5'3'5'replicationfork•ManyDNAfragmentsaresynthesizedsequentiallyontheDNAtemplatestrandhavingthe5´-end.TheseDNAfragmentsarecalledOkazakifragments.Theyare1000–2000ntlongforprokaryotesand100-150ntlongforeukaryotes.•ThedaughterstrandconsistingofOkazakifragmentsiscalledthelaggingstrand.OkazakifragmentsContinuoussynthesisoftheleadingstrandanddiscontinuoussynthesisofthelaggingstrandrepresentauniquefeatureofDNAreplication.Itisreferredtoasthesemi-continuousreplication.Semi-continuousreplicationSection2EnzymologyofDNAReplicationEnzymesandproteinfactorsproteinMr#functionDnaAprotein50,0001recognizeoriginDnaBprotein300,0006opendsDNADnaCprotein29,0001assistDnaBbindingDNApolElongatetheDNAstrandsDnaGprotein60,0001synthesizeRNAprimerSSB75,6004single-strandbindingDNAtopoisomerase400,0004releasesupercoilconstraint•ThefirstDNA-dependentDNApolymerase(shortforDNA-polI)wasdiscoveredin1958byArthurKornbergwhoreceivedNobelPrizeinphysiologyormedicinein1959.§2.1DNAPolymeraseDNA-polofprokaryotes•Later,DNA-polIIandDNA-polIIIwereidentifiedinexperimentsusingmutatedE.colicellline.•Allofthempossessthefollowingbiologicalactivity.1.53polymerizing2.exonucleaseDNA-polofE.coliDNA-polI•Mainlyresponsibleforproofreadingandfillingthegaps,repairingDNAdamageKlenowfragment•smallfragment(323AA):having5´→3´exonucleaseactivity•largefragment(604AA):calledKlenowfragment,havingDNApolymerizationand3´→5´exonucleaseactivityNendCendcaroidDNA-polⅠDNA-polII•TemporaryfunctionalwhenDNA-polIandDNA-polIIIarenotfunctional•Stillcapablefordoingsynthesisonthedamagedtemplate•ParticipatinginDNArepairingDNA-polIII•Aheterodimerenzymecomposedoftendifferentsubunits•Havingthehighestpolymerizationactivity(105nt/min)•ThetrueenzymeresponsiblefortheelongationprocessStructureofDNA-polIIIα：has5´→3´polymerizingactivityε：has3´→5´exonucleaseactivityandplaysakeyroletoensurethereplicationfidelity.θ:maintainheterodimerstructureDNA-polofeukaryotesDNA-pol:elongationDNA-polIIIDNA-pol:initiatereplicationandsynthesizeprimersDnaG,primaseDNA-pol:replicationwithlowfidelityDNA-pol:polymerizationinmitochondriaDNA-pol:proofreadingandfillinggapDNA-polIrepairing§2.2Primase•AlsocalledDnaG•Primaseisabl