分子生物学-ch-4-DNA-replication

Chapter4DNAreplicationI.Semi-conservativeandsemi-discontinuous1.Semi-conservativereplication(半保留复制)1958年Meselson-Stahl实验先将大肠杆菌放在15NH4C1培养基中生长15代，使几乎所有的DNA都被15N标记后，再将细菌移到只含有14NH4C1的培养基中培养。随后，在不同的时间取出样品，用十二烷基硫酸钠(SDS)裂解细胞后，将裂解液放在CsC1溶液中进行密度梯度离心(140000g，20小时)。离心结束后，从管底到管口，溶液密度分布从高到低形成密度梯度。DNA分子就停留在与其相当的CsC1密度处，在紫外光下可以看到形成的区带。TheMeselson-StahlExperimentAutoradiograph，放射自显影2.Semi-discontinuousreplication半不连续复制前导链(leadingstrand)滞后链(laggingstrand)冈崎片段(Okazakifragment)II.DNApolymeraseDNA聚合酶Template+primer+4dNTPDNApolymerasedsDNADirection:5’3’ThedirectionofsynthesisofthenewDNAchainisalwaysfrom5’to3’.5’to3’AdditionofMonomersAtthegrowingendoftheDNAchain,phosphodiesterbondformationbetweenthe3’-OHofthelastnucleotideandthe5’-phosphateoftheincomingdNTP.TosynthesizeachainofDNA,DNApolymeraserequiresPrimer:providingafree3’-OHgroup;Template:DNApolymerasetakeinstructionsfromtemplatesdNTPs:dATP,dGTP,dTTP,dCTP1.DNApolymeraseIDNApolymeraseⅠisa103-kdsinglepolypeptidechain.DNAPolI’sfunctioninreplication:1.RemovesRNAprimer(5’→3’exonucleaseactivity)2.Fillingthegapbetweenthefragments(5’→3’polymerizationactivity)PolⅢ填充小缺口（几个碱基）;PolI能填充大缺口.2.DNAPolymeraseIIIPolIIIisacomplexenzymewithmanysubunits.CatalyticCorecomposedofαεθsubunits.ItisthereplicaseinE.coli.Subunitsinthecatalyticcoreα:~130kdgene:dnaE主要具有聚合酶活性ε:~27.5kdgene:dnaQ(3’→5’外切酶活性)θsubunit:~10kdgene:holEβ:progressivity保证进行性ADNApolIIIholoenzymecontainsatleast20subunits.β亚基起着滑动钳夹的作用，使PolⅢ与DNA保持在一起。ProofreadingAllthreeE.coliDNApolymeraseshas3’→5’exonucleaseactivity:therateofmis-incorporation:basedonthedynamicstructureofthebasesshouldbe~10-5,in-reality:therateofthemis-incorporationislessthen10-9BacterialDNApolymerasesI,IIandIIIpolI–mostabundant(400/cell)andverystable–RNAprimerremoval,polymerase,exonucleasepolII–unknownabundance–Polymerase,exonuclese,DNArepair?polIII–lowabundance(15/cell)–Polymerase,exonuclease,DNAreplicationPolIIIisverycomplex,manysubunitsIII.OtherenzymesandproteinsinvolvedinDNAreplicaion1.DNAPrimaseDNApolymerasesrequiresforafree3’-OH:1.DNAprimasecatalyzesthesynthesisofshort(10-60nt)RNAstrandsthatarecomplementarytothetemplatestrands.2.DNApolymeraseIIIthenusesthefree3’-OHoftheRNAprimertoextendthechainsbyadditionofdNTP.2.DNAhelicase&SSBproteinsDNAhelicase:catalyzestheunwindingoftheparentaldoublehelixSingleStrandBindingprotein(SSB):keeptheunwoundstrandsinanextendedformforreplicationDNAhelicase&SSBproteins3.TopoisomeraseProducestransientsingle-strandbreaksinDNA1.ActasanaxesofrotationorswivelsduringDNAreplication2.RemovessupercoilsfromDNAoneatatimeTopoisomeraseI(拓扑异构酶I)4.DNALigase•DefinitionofDNAligase:Anenzymethatclosesnicksordiscontinuitiesinonestrandofdouble-strandedDNAbycreatinganesterbondbetweenadjacent3'OHand5'PO4endsonthesamestrand•JoiningofadjacentDNAfragments•DNAlaggingstrandsynthesisisdiscontinuous:theOkazakifragmentsPrimaseHelicaseTopoisomeraseSSBModelforDNAreplicationActionattheReplicationFork:TwoPolymerasesactasaDimerIV.DynamicsofDNAreplicaion起始(initiation)延伸(elongation)终止(termination):发生在母代分子复制完成时。Example1.E.coli(1)InitiationDenovoinitiation从头起始BacterialDNAReplicationbeginsataSingleOriginandProceedsBidirectionallyOriginofReplicationInE.coli，thereisonlyoneoriC（1）含有多个短重复序列，（2）A-Trich,（3）能被专一性的起始蛋白识别，形成replisome(复制体).Replicationorigin复制起始点Bidirectional&Semi-discontinuousθ复制环状DNA复制时状如θ，称θ复制，又叫Cairns复制。复制中间体称为Cairns分子。primosome•Inmolecularbiology,aprimosomeisaproteincomplexresponsibleforcreatingRNAprimersonsinglestrandedDNAduringDNAreplication.•Theprimosomeconsistsofsevenproteins:DnaGprimase,DnaBhelicase,DnaChelicaseassistant,DnaT,PriA,PriB,andPriC.TheprimosomeisutilizedonceontheleadingstrandofDNAandrepeatedly,initiatingeachOkazakifragment,onthelaggingDNAstrand.(2)ElogationReplisome:thecomplexofkeyreplicationproteinswithDNAatthereplicationforkRecentpapersshedinsightintothearchitectureanddynamicsofthecomponentsofthebacterialreplisome.HelicaseGyrasePrimaseDNApolymeraseIIIholoenzymeDNApolymeraseILigase(3)Termination复制终止的序列ter与TUS蛋白结合initiation共价延伸主要指滚环复制（也称σ复制）；先导链基于一亲链基础上共价延伸。Example2:ΦX174