Ch3.DNAReplication,Repair3.1.复制的特点:1.复制机制的多样性;方式多样:线状、环状D-环复制,θ型复制,滚环复制2.复制的完整性;3.DNA复制与细胞周期的协调性(一致)4.DNA复制的真实性DNA复制错误率10-9—10-10(生物进化、多样性的来源)ThemammaliancellcycleG1SG2MG0DNAsynthesisandhistonesynthesisGrowthandpreparationforcelldivisionRapidgrowthandpreparationforDNAsynthesisQuiescentcellsphasephasephasephaseMitosis3.2.DNA复制反应的共同特点1.半保留复制;以母链为模板,合成新的互补链2.半不连续复制;3.有特定的复制起点;复制子(复制单元)4.需要引物;(3’-OH)5.复制真实性;6.复制叉的双向移动,DNA合成的单向延伸5’→3’;8.复制受控制DNAreplicationissemi-conservativeParentalDNAstrandsDaughterDNAstrandsEachoftheparentalstrandsservesasatemplateforadaughterstrandDNA的半不连续复制由于DNA的两条链为反向平行,以复制叉移动的方向为基准,一条链为3’→5’,其新合成的DNA链沿5’→3’连续进行——前导链另一条链为5’→3’,新生的DNA链先合成短的冈崎片段,不连续合成——滞后链,再由DNA连接酶连接成完整的DNA链。这种前导链的连续复制和滞后链的不连续复制称为半不连续复制。DiscontinuoussynthesisofDNA3’5’5’3’3’5’BecauseDNAisalwayssynthesizedina5’to3’direction,synthesisofoneofthestrands...5’3’...hastobediscontinuous.Thisisthelaggingstrand.5’3’3’5’5’3’RNAprimer5’3’3’5’3’5’directionofleadingstrandsynthesisdirectionoflaggingstrandsynthesisreplicationfork5’3’5’3’MovementofthereplicationforkMovementofthereplicationforkRNAprimerOkazakifragmentRNAprimer5’从复制起点到终点的DNA单位——复制子(复制单元)originsofDNAreplication(every~150kb)replicationbubbledaughterchromosomesfusionofbubblesbidirectionalreplicationOriginsofDNAreplicationonmammalianchromosomes5’3’3’5’5’3’3’5’3’5’5’3’3.3.原核生物DNA的复制1.参与DNA复制的酶和蛋白因子螺旋酶(Helicase)促使DNA在复制叉处打开双链,与单链DNA结合,与ATP结合,分解成ADP+Pi,产生能量沿DNA链向前运动,促使DNA双链解开。单链DNA结合蛋白(SSBP)与单链DNA结合,防止解开的单链重新配对形成双链或被核酸酶降解;使单链DNA呈伸展状态,无弯曲和结节,利于作为模板;SSBP可重复使用。——形成稳定的单链构象拓扑异构酶Topoisomerase——导入负超螺旋引物酶Primase催化引物RNA分子的合成DNA聚合酶DNAPolymerase特点:1.以dNTP为前体催化合成DNA;2.需要模板和引物;3.催化dNTP加到DNA链的3’-OH端;4.催化合成方向5’→3’。PropertiesofDNApolymerasesDNApolymerasesofE.coli_polIpolIIpolIII(core)Polymerization:5’to3’yesyesyesProofreadingexonuclease:3’to5’yesyesyesRepairexonuclease:5’to3’yesnonoDNApolymeraseIIIisthemainreplicatingenzymeDNApolymeraseIhasaroleinreplicationtofillgapsandexciseprimersonthelaggingstrand,anditisalsoarepairenzyme•allDNApolymerasesrequireaprimerwithafree3’OHgroup•allDNApolymerasescatalyzechaingrowthina5’to3’direction•someDNApolymeraseshavea3’to5’proofreadingactivityDNApolymerasesIIIDNA连接酶将双链DNA上的切口连接起来;主要用于冈崎片段的连接,DNA修复、重组,两个复制单元间片段的连接。连接双链DNA的要求:切口3’-OH5’-磷酸基团需要能量newlysynthesizedDNA(Okazakifragment)5’3’5’3’DNAligasesealsthegapbycatalyzingtheformationofa3’,5’-phosphodiesterbondinanATP-dependentreaction2.大肠杆菌DNA的复制过程起始——延伸——终止DNA螺旋酶解开双链,拓扑异构酶形成负超螺旋SSBP结合单链DNA复制因子组装成引发前体RNA引物合成在DNApolIII作用下延伸引物酶引发体起始阶段:dnaA蛋白+重复序列(9bp)→dnaB和dnaC+重复序列(13bp)结合→其它蛋白因子结合——装配成全酶(引发前体)+引物酶→引发体PrimosomeInitiationofDNAsynthesisattheE.coliorigin(ori)5’3’3’5’originDNAsequencebindingofdnaAproteinsAAAdnaAproteinscoalesceDNAmeltinginducedbythednaAproteinsAAAAAAAAAAAABCdnaBanddnaCproteinsbindtothesingle-strandedDNAdnaBfurtherunwindsthehelixAAAAAABCdnaBfurtherunwindsthehelixanddisplacesdnaAproteinsGdnaG(primase)binds...AAAAAABCG...andsynthesizesanRNAprimerRNAprimerBCG5’3’templatestrandRNAprimer(~5nucleotides)PrimasomednaB(helicase)dnaCdnaG(primase)OH3’5’3’5’3’RNAprimernewlysynthesizedDNA5’5’DNApolymerase链的延伸•螺旋酶解开双链;•拓扑异构酶改变双螺旋数,使复制叉前进;•SSBP结合,保持双链状态;•DNA聚合酶Ⅲ合成前导链和滞后链,前导链引发1次,滞后链引发几次;•DNA聚合酶Ⅰ切除RNA引物,补齐空缺的碱基;•DNA连接酶连接冈崎片段。5’3’3’5’ProteinsatthereplicationforkinE.coliRepprotein(helicase)Single-strandbindingprotein(SSB)BCGPrimasomepolIpolIIIpolIIIDNAligaseDNAgyrase-thisisatopoisomeraseII,whichbreaksandresealstheDNAtointroducenegativesupercoilsaheadofthefork3’RNAprimer5’DNApolymeraseIIIinitiatesattheprimerandelongatesDNAuptothenextRNAprimer5’5’3’5’newlysynthesizedDNA(100-1000bases)(Okazakifragment)5’3’DNApolymeraseIinititatesattheendoftheOkazakifragmentandfurtherelongatestheDNAchainwhilesimultaneouslyremovingtheRNAprimerwithits5’to3’exonucleaseactivitypolIIIpolInewlysynthesizedDNA(Okazakifragment)5’3’5’3’DNAligasesealsthegapbycatalyzingtheformationofa3’,5’-phosphodiesterbondinanATP-dependentreaction终止环状DNA:合成完即终止;线状DNA:末端合成问题。ComponentsofthereplicationapparatusdnaAbindstooriginDNAsequencePrimasomednaBhelicase(unwindsDNAatorigin)dnaCbindsdnaBdnaGprimase(synthesizesRNAprimer)DNAgyraseintroducesnegativesupercoilsaheadofthereplicationforkRepproteinhelicase(unwindsDNAatfork)SSBbindstosingle-strandedDNADNApolIIIprimaryreplicatingpolymeraseDNApolIremovesprimerandfillsgapDNAligasesealsgapbyforming3’,5’-phosphodiesterbond复制体:在DNA复制过程中,在复制叉附近,形成以两套DNA聚合酶Ⅲ全酶分子、引发体和螺旋酶构成的类似核糖体大小的复合体称为DNA复制体。回环模型:在DNA复制叉处由两套DNA聚合酶在同一时间分别复制前导链和滞后链,如果滞后链模板环绕DNA聚合酶Ⅲ全酶,并通过DNA聚合酶Ⅲ,再回折与未解链的双链DNA在同一方向,则滞后链的合成与前导链的合成在同一方向上进行。ReplisomemodelReplisomeincovalenceelongationSSB3.DNA复制方式:θ型复制:滚环复制许多病毒、细菌因子的复制方式D-环复制:线粒体、叶绿体环状DNA复制方式之一;特点:1.以轻链为模板先复制其互补链;2.以轻链为模板的子链继续合成,以重链为模板开始合成;3.复制完成。两条链的合成不是同步的D—环复制4.线状DNA的复制线状DNA复制的一个重要问题是末端RNA引物消除后如何补齐?①形成环状DNA进行复制λ-phage②形成聚合体T7DNA③末端形成发夹结构痘病毒线状DNA的不完全复制带发夹环的线性病毒DNA的复制④不需要RNA引物的复制腺病毒蛋白质分子介入,进行末端的引发。腺病毒中发现80Kd的末端结合蛋白(Tp),由丝氨酸残基(Ser)的-OH基团通过磷酸二酯键与胞苷酸(C)的5’-共价连接。Tp内的胞苷酸与线状DNA的3’端配对,成为病毒DNA复制起始的引物,在酶作用下合成新链。其它枯草杆菌噬菌体φ29中也发现了27Kd的末端蛋白。ReplicationoflinearAdnovirus-2DNA35937bppTPCGIR103-162IR;including50bpreplicationoriginrichAT&1thC/Ghig