生物化学二-dna biosynthesis and replication zhanglan复旦生物医学研究院

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

daughter7FourCharacteristicsofSemi-conservativeBidirectionalSemi-discontinuousHighProposedModelsofDNAInthelate1950s,threedifferentmechanismswereproposedforthereplicationofDNADNAsemiconservative半保 （semiconservative即新的双链DNA中，一股链来自模板，一股为新合成半保 的意整无误的传递给子代，体现了遗传的保守半保 实验依1958年M.Messelson等用实验以了证双螺旋结构是半保 的分基SemiconservativemodelofDNA++++-RandomornonrandomseparationincellHowHowistheCGmethylationmaintaintedduringDNAreplication?DNAsynthesisbeginsatasitetermedoriginofSynthesisofDNAproceedsaroundthebacterial 裂解的细胞曝露在感光乳胶后，发现在伸长的的点出发同时向两个方向。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

能在未确定前一个核苷Abletolink

DNAPrimerOnLagging对功能，所以不需要引物从新开始合成错配可能性大，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修复(应急状态DNA-pol有从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双链DNA3'突出(3'overhang)的打平；Structureresemblesletthroughthepalm;聚合酶III ——主要 酶，兼有校读、纠错的功有从5’——3’延伸多核苷酸链的聚合酶活性，有模板依赖性，其延伸的方磷酸二酯键连接，同时释出一个PPi。DNA聚合酶延伸多核苷酸链的方向总是5’至3’有从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