生物化學(xué)二-dna biosynthesis and replication zhanglan復(fù)旦生物醫(yī)學(xué)研究院

GeneticInformationContentsofThis?TheCharacteristicsofDNA?TheDNAReplicationOtherReplication3 ?SomethingneedsofDNACentral miRNAandsiRNAinducegenencRNAsuppressgeneReplicationDNAhastobecopiedbeforeacellDNAiscopiedduringtheSorsynthesisphaseofinterphase.NewcellswillneedidenticalDNA8SynthesisSphaseduringinterphaseofthecellNucleusof9GeneralConceptsOfDNAAreactioninwhichdaughterDNAsaresynthesizedusingtheparentalDNAsasthetemplate.Transferringt eticinformationtothedescendantgenerationwithahighfidelityparental

daughter7FourCharacteristicsofSemi-conservativeBidirectionalSemi-discontinuousHighProposedModelsofDNAInthelate1950s,threedifferentmechanismswereproposedforthereplicationofDNADNAsemiconservative半保 （semiconservative即新的雙鏈DNA中，一股鏈來自模板，一股為新合成半保 的意整無誤的傳遞給子代，體現(xiàn)了遺傳的保守半保 實驗依1958年M.Messelson等用實驗以了證雙螺旋結(jié)構(gòu)是半保 的分基SemiconservativemodelofDNA++++-RandomornonrandomseparationincellHowHowistheCGmethylationmaintaintedduringDNAreplication?DNAsynthesisbeginsatasitetermedoriginofSynthesisofDNAproceedsaroundthebacterial 裂解的細胞曝露在感光乳膠后，發(fā)現(xiàn)在伸長的的點出發(fā)同時向兩個方向。withtwoforksReplicationforksvisibleinSemi-discontinuousreplicationreplicationreplication OkazakiOkazakileadingleading

Directionofonleading

1000–2000ntinprokaryotes,100-150ntinTwodimensionalviewofareplicationHighDNApolymeraseinitiallymakesabout1in10,000basepairingerrors(10-4-10-5)EnzymesproofreadandcorrecttheseThenewerrorrateforDNAthathasbeenproofreadis1in1billionbasepairingerrors(10-8-10-10)TheapplicationofDNAsynthesisinCloningDifferentDNApolymerase,DifferentterminalofPCRproducts3’1nucleotideBluntReplicationReplicationProcessandComponentFourComponentsofDNAreplication

doublestrandedDNAshortRNAfragmentwithafree3′-OHendpolymerase(DDDP),otherenzymes,protein8Enzymesandprotein#DnaA1recognizeDnaB6openDnaC1assistDnaBDNAElongatetheDnaG1synthesizeRNA4single-strandDNA4releasesupercoilSequentialInitiation:recognizethestartingpoint,separatedsDNA,primersynthesis,…Elongation:adddNTPstotheexistingstrand,formphosphoesterbonds,correctthemismatchbases,extendingtheDNAstrand,…Termination:stoptheReplicationofTheOriginofTheoriginofreplicationinE.coliistermedDnaA–originofDnaADnaDnaDnaDna DNAdoublehelixaretightlycoupled.HightemperatureisneededtobreakthemSSBSSB

UsesenergyfromATPtounwindtheduplex

SSBSSBproteinmaintainstheDNAtemplateinthesinglestrandformintopreventthedsDNAformation;protectthevulnerablessDNAfromDNAPolymeraseCannotInitiatenewUnabletocovalentlythe2

能在未確定前一個核苷Abletolink

DNAPrimerOnLagging對功能，所以不需要引物從新開始合成錯配可能性大，要去除，取而代之DNADNA-polofThefirstDNA-dependentDNApolymerase(shortforDNA-polI)wasdiscoveredin1958byArthurKornbergwhoreceivedNobelPrizeinphysiologyormedicinein1959.DNA聚合酶DNA聚合酶DNA聚合酶1103889005′→3′核酸聚合+++3′→5′核酸外切酶+++5′→3′+--115000~60≥500修復(fù)(應(yīng)急狀態(tài)DNA-pol有從5’——3’外切酶的活性，其作用是切除RNA引物 N

KlenowDNA-pol C smallfragment(323AA):having5′→3′exonucleaselargefragment(604AA):calledKlenowfragment,DNApolymerizationand3′→5′exonuclease雙鏈DNA3'突出(3'overhang)的打平；Structureresemblesletthroughthepalm;聚合酶III ——主要 酶，兼有校讀、糾錯的功有從5’——3’延伸多核苷酸鏈的聚合酶活性，有模板依賴性，其延伸的方磷酸二酯鍵連接，同時釋出一個PPi。DNA聚合酶延伸多核苷酸鏈的方向總是5’至3’有從3’——5’DNA-pol5′→3′exonucleasecutRNAprimerorexcisemutated

DNA-polI，III3′→5′excisemismatched 3' Proofreadingbythe3’→5’exonucleaseactivityofDNApolymerasesduringDNAreplication.Question:Question:whatfactorsmakesurethatonlytheprimerbutnottheDNAisdigestedbyPolI?Thingsneedtobedoneafterelongation DNAPolI:digestRNA

DNAPolI：fillthe

DNADNATusfactorsrecognizetheterminalThereplicationofE.coliisbidirectionalfromoneorigin,andthetworeplicationforksmustmeetatonepointcalledter.Alltheprimerswillberemoved,andallthefragmentswillbeconnectedbyDNA-polIandligase.BacterialDNAreplication----oneorigin,twoDnaGModelofreplicationDnaGDnaDnaADnaDnaCDnaBExperiment1HowtoprovethattheOkazakifragmentisoneortwonucleosomeslength?12Whatisthemodelofoctamer)assemblyafterDNAreplication?21

WhycelluseRNAprimerduringDNAreplication?2HowtheDNAmethylationcontrolthereplication?Whathappenedinthetumorcell?23WhichfactorsdeterminetheRNAintegratedinthehostgenome?34WhatwillhappeniftheRNAprimersnotdegratedduringDNAreplication?4ReplicationofDNAreplicationiscloselyrelatedwithcellcycle.Multipleoriginsononechromosome,andreplicationsareactivatedinasequentialorderratherthanDifferentregionsreplicatedatdifferentReplicationofeukaryotes---multipleorigins,two fusionof 5' TheDNAreplicationinitiatedattheterminusincellslackingRecGistheresultofaPriA–PriB-mediatedloadingofDnaBatabranchedDNAstructuregeneratedinthisregionshelterinmultiproteincomplexesprotect omericDNAends. edeprotected，aspecializedformofDNAdamageresponseistriggered.omericDNAdamageresponseinduceG1arrest.omericDNAdamageresponserequiresp53fortheG1arrestandthemaintenanceofgenomicDNApolymerasescanonlysynthesizeDNAonlyinthe5’to3’directionandcannotinitiateDNAsynthesisThesetwofeaturesposeaproblematthe3’endoflinearchromosomesTheeukaryoticcellsuse omerasetomaintaintheintegrityofDNA omeraseiscomposedofomeraseRNAomerasereverseItisabletosynthesizeDNAusingRNAasthetemplate.withRNAtemplatetobindtoDNAstrandsomeraseanditsRR 1bindstoPCNA,aninteractionthatwasimportantfornormalDNAreplication,replicationforkstability,and omerestability.OtherOtherReplicationReverse containing+ssRNAgenomeSynthesisofssDNAcomplementarytossRNA,formingaRNA-DNAhybrid.HydrolysisofssRNAintheRNA-DNAhybridbyRNaseactivityofreversetranscriptase,leavingssDNA.SynthesisofthesecondssDNAusingtheleft