Part-II-23-重组.ppt

PartIIDNA复制、突变、损伤和修复、重组和转座,2.1DNA复制的酶学和机制2.2DNA的突变、损伤和修复2.3同源重组、位点特异性重组及转座作用,可遗传的变异:,突变（点突变，染色体变异）,DNA分子结构的永久性改变,DNA重组(Recombination),发生在DNA分子内或DNA分子之间核苷酸序列的交换、重排和转移现象，是已有遗传物质的重新组合过程。,Roles,Generatingnewgene/allelecombinations(crossingoverduringmeiosis)Generatingnewgenes(e.g.,IgGrearrangement)IntegrationofaspecificDNAelementDNArepair,2.3.1同源重组,HomologousRecombination,分子间,分子内,一种在两个DNA同源序列之间直接进行交换的重组形式。,occurbetweensequencesthatarenearlyidentical,involvesareciprocalexchangeofsequencesofDNA,e.g.betweentwohomo-chromosomesthatcarrythesamegeneticloci.,largefragmentexchange,Recombinationsiteisinhotspotmostly,Recombinasebeneeded(RecA,BCD),一.同源重组的分子模式,1.Hollidaymodel(RobinHolliday,1964),2个同源DNA分子靠近,各有一个DNA单链被切开,分别侵入另一个DNA分子,Holliday中间体,拆分,AligntwohomologousDNAmolecules,NicktheDNAatthesameplaceonthetwomolecules,Exchangestrandsandligate,TheintermediatethatisformediscalledaHollidayintermediateorHollidaystructure.,onemoleculeisnowrotatedthrough180withrespecttotheother,Resolvethestructure,Thebasic(simple)model,Noncrossoverrecombination,Crossoverrecombination,Amorerealisticmodel,Branchmigration.,patch,Noncrossoverrecombination(withheterologousDNA),Crossoverrecombination,branchmigration,Figure19-9.Aprototypemechanismforgeneticrecombination.(a)Twohomologousdoublehelicesareshown.Eachpairrepresentsachromatidandthetwopairsrepresenttwononsisterchromatids.Thehelicesarealignedsothatthebottomstrandofthefirsthelixhasthesamepolarityasthetopstrandofthesecondhelix.(b)Twoparallelortwoantiparallelstrandsarecut.(c)Thefreeendsbecomeassociatedwiththecomplementarystrandsinthehomologousdoublehelix.(d)Ligationcreatespartlyheteroduplexdoublehelices,theHollidaystructure.(e)Migrationofthebranchpointisbycontinuedstrandtransferbythetwopolynucleotidechainstakingpartinthecrossover.(f)Resolutioncanoccurinoneoftwoways,whichwillbedescribedindetaillater.(AfterH.PotterandD.Dressler,ColdSpringHarborSymposiumonQuantitativeBiology43,1979,970.ColdSpringHarborLaboratory,ColdSpringHarbor,NY.),2.theMeselson-Raddingmodel(MattMeselsonandCharledRadding,1975),单链断裂,3.Double-strand-breakmodel(Szostak1983),双链断裂,二.参与同源重组的主要蛋白,e.g.E.Coli.,RecARecBCDRuvARuvBRuvC,RecA,Monomer:352Aa,38KDa,含有两个DNA结合位点（分别结合单链DNA和双链DNA);,促进2个DNA分子的链交换,ThreestagesofparticipationofRecAinstrandexchange,Presynapsis:RecAandSSBcoatthessDNA;Synapsis:alignmentofcomplementarysequencesinthessDNAanddsDNA;Postsynapsis:strandexchange.,RecBCD由RecB，RecC，RecD三个亚基组成；,DNAhelicaseactivityexonucleaseactivity(exonucleaseV)endonucleaseactivityATPaseactivity,具有多酶活性,1.RecBCDproteinnicksonestrandtothe3-sideofthechisite.,2.TheDNAhelicaseactivityofRecBCDthenunwindstheDNAthroughthechi-siteformingasingle-strandtail.,3.RecAandSSBcoatthetail.,4.RecApromotesinvasionofanotherDNAduplex,formingaD-loop.,5.RecAhelpstheinvadingstrandscanforaregionofhomologyintherecipientDNAduplex.,6.Theinvadingstrandbase-pairedwithahomologousregion,releasingSSBandRecA.,7.RecBCDnicksthelooping-outstrand.RecAandSSBhelpsstrandexchange.,Chi位点5GCTGGTGG33CGACCACC5,RecBCD可能提供一条含有游离3端的单链区域,RuvA,tetramer,识别Holliday连接点,RuvB,hexamerring,具有解旋酶和ATPase活性,RuvA和RuvB催化branchmigration,RuvB,RuvA,Directionofbranchmigration,awayfromRuvB,RuvC（resolvase),dimer,核酸内切酶,特异的作用序列5（A/T)TT(G/C)3,RuvC能拆分Holliday中间体,三.同源重组的分子机制,1.RecBCDproteinnicksonestrandtothe3-sideofthechisite.,2.TheDNAhelicaseactivityofRecBCDthenunwindstheDNAthroughthechi-siteformingasingle-strandtail.,3.RecAandSSBcoatthetail.,4.RecApromotesinvasionofanotherDNAduplex,formingaD-loop.RecAhelpstheinvadingstrandscanforaregionofhomologyintherecipientDNAduplex.,5.Theinvadingstrandbasepairedwithahomologousregion,releasingSSBandRecA.,6.RecBCDnicksthelooping-outstrand.RecAandSSBhelpsstrandexchange.,8.Branchmigrationoccours,sponsoredbyRuvAandRuvB.,9.RuvCresolvesthestructure.,7.ThenicksaresealedbyDNAligase,yieldingaHollidayjunction.,四.真核生物的同源重组,重组发生在第一个减数分裂的前期,五.通过同源重组进行基因敲除,2.3.2位点特异性重组,Site-specificRecombination,发生在DNA特异性位点上的重组。,Asthenameimplies,thistypeofrecombinationinvolvestheexchangeofgeneticmaterialatveryspecificsitesonly.RequireslesssequencehomologybetweenrecombiningDNAsthandoeshomologousrecombination.Specializedproteinsareneededtorecognizethesesitesandtocatalyzetherecombinationreactionatthesesites.,Thestepsandfeatures,StrandexchangeFormationofahollidayintermediateBranchmigrationResolution,betweentwoDNAmoleculars,withinoneDNAmolecular,Invertedrepeats,Directrepeats,Thestructureofrecombinaserecognitionsite,Invertedrepeats,Directrepeats,e.g.噬菌体DNA整合到E.ColiDNA,att(attachmentsite),E.ColiattB,BOB,phageattP,POP,Int(integrase),IHF(integrationhostfactor),30bp,255bp,Xis(excisionase),Invertedrepeats,Site-specificintegrationofl,Invertedrepeats,Directrepeats,MechanismofIntaction,系列转酯反应,2.3.3转座重组,TranspositionRecombination,DNA上的一段核苷酸序列从一个位置转移到另一个位置的现象。,转座子（transposons)：发生转位的DNA片断,BarbaraMcClintockspentmanyyearsstudyingthebehaviorofunusualgeneticelementsinmaize.Sheconcludedthattheseelementswere,infact,mobile.Herwork,allthemoreamazingbecausemuchofitwascarriedoutbeforethestructureofDNAwassolved,waslargelyignoreduntilthemid1970swhensimilarelementswerediscoveredinbacteria.,1947,一.原核生物的转座子,（一）IS(insertionsequence),InsertionSequencesorISelementsarethefirsttransposonsdiscoveredinbacteria.thesimplestmobileelementconsistofafairlyshort(700-1800bp)DNAsegmentflankedbya10-40bpinvertedrepeatsequence.Thesegmentcodesfortheprotein(transposase,tnpA)thatcatalysesthetranspositionevent.,（二）complextransposon,l两侧具有IR(3540bp),l内部含有转座酶基因（tnpA）、解离酶基因（tnpR）、抗生素抗性基因。,（三）compositetransposon,2个IS插入到功能基因（抗生素或其它毒性抗性基因）两端，形成复合转座因子。,每个IS可以独立转位，也可以与插入的功能基因作为整体一起转位。,TheISelementsmaybeinthesameorintheoppositeorientationwithrespecttooneanother.,（四）BacteriophageElements(Mu),38kblinerdsDNA;WithoutIRelementsintwosides;OnlygeneAandgeneBinvolvedintransposition;geneAcodefortransposase;,二.原核生物的转座机制,ReplicativetranspositionTheelementreplicatesitselfandonecopyremainsatitsoriginalsite.,NonreplicativetranspositionTheoriginalelementisexcisedfromtheoriginalsiteleavingadouble-strandbreakbehind.Cut&PasteModel,Replicativetransposition,Nonreplicativetransposition,Staggeredcut,转座机制replicatingtranspositioninprokaryote,Tn,target,Strandstransfer,copyingofTn&targetseq.,cointegrater&resolution,Conclusion,c)转座子的IR序列是转座酶的重要识别位点，与转座，切除有关,a)转座过程是由Donor提供Tncopy到targetsite，涉及酶切、复制、重组的遗传学过程。,b)转座完成后，在Tn的两端出现targetsite序列的正向重复，其长度取决于staggeredcutting的长度。,d)Cointegrater是转座过程的中间体(具有两个Tn和两个targetsite),其稳定性依Tn不同而异；最后通过resolution完成转座过程.,三.真核生物的转座子,1947BarbaraMcClintock,玉米糊粉层花斑不稳定现象伴随的遗传事件,玉米的Ac-Ds系统,Ac（activatorelement),Ds（dissociationelement),Ac:自主型（autonomous)转座子,编码转座所需的酶，能独立地进行转座；,Ds:非自主型（non-autonomous)转座子，不能独立地进行转座，但在相应的转座酶作用下可以转座。,果蝇的Pelement,P元件中的转座酶基因表达通常处于抑制状态。,P元件只在P型雄蝇与M型雌蝇杂交后代的性细胞