分子生物學(xué)課件：ch10 Homologous Recombination at the Molecular Level

Chapter10HomologousRecombinationattheMolecularLevelChapter10DNA分子內(nèi)或分子間發(fā)生遺傳信息的重新組合，稱為遺傳重組

(geneticrecombination),或者基因重排(generearrangement)。重組產(chǎn)物為重組體DNA(recombinantDNA)。DNA的重組廣泛存在于各類生物,以允許遺傳交換、染色體上基因重排、斷裂DNA及停滯復(fù)制叉的修復(fù)。真核生物基因間重組多發(fā)生在減數(shù)分裂(meiosis)時同源染色體之間的交換(crossover)。DNA重組不僅對生物進化起著關(guān)鍵的作用，還參與許多重要的生物學(xué)過程。它為DNA損傷或復(fù)制障礙提供修復(fù)機制。某些生物的基因表達(dá)受DNA重組的調(diào)節(jié)。DNA重組包括同源重組（homologousrecombination）、位點專一性重組（site-specificrecombination）和轉(zhuǎn)座重組（transpositionrecombination）等類型。GeneticRecombinationChapter10HomologousrecombinationisatypeofgeneticrecombinationinwhichnucleotidesequencesareexchangedbetweentwosimilaroridenticalmoleculesofDNA.ItismostwidelyusedbycellstoaccuratelyrepairharmfulbreaksthatoccuronbothstrandsofDNA,knownasdouble-strandbreaks.HomologousRecombination同源重組（HomologousRecombination)是指發(fā)生在姐妹染色單體（sisterchromatin)之間或同一染色體上含有同源序列的DNA分子之間或分子之內(nèi)的重新組合。

Chapter10同源重組的特點：1）要求較大的同源DNA片段（>75bp）才能進行交換；同時需要重組酶（如RecA）參與。2）同源片段相同或相近，在任何位點均可發(fā)生重組，但是存在重組熱點；3）整個基因組重組頻率不恒定，受綜合效應(yīng)和局部效應(yīng)的影響；卵母細(xì)胞>精母細(xì)胞。女性聯(lián)會復(fù)合體的長度及重組頻率都是男性的2倍。同源重組的功能：1)維持生物種群的多樣性；2）染色體瞬間的物理連接，對染色體正確分離到子代細(xì)胞中至關(guān)重要。3）用于損傷修復(fù)。HomologousRecombination同源重組是最基本的DNA重組方式，由一系列特異的酶所催化并通過鏈的斷裂和再連接，在兩個DNA分子同源序列間進行單鏈或雙鏈片段的交換。

Chapter10Chapter10OUTLINEModelsforHomologousRecombination(twomodels)**HomologousRecombinationProteinMachines(RecBCDpathway)**HomologousRecombinationinEukaryotes(meiosisrecombination,RecA-likeproteins)Mating-TypeSwitching(ageneconversionevent)GeneticConsequencesoftheMechanismofHomologousRecombination(exchangeorconversion)Chapter10Topic1:ModelsforHomologousRecombination

HollidayModel(Holliday模型)Double-StrandBreak(DSB)RepairModel(雙鏈斷裂修復(fù)模型)Chapter10AlignmentoftwohomologousDNAmolecules.（聯(lián)會/排列整齊）IntroductionofbreaksintheDNA（斷裂）FormationofinitialshortregionsofbasepairingbetweenthetworecombiningDNAmolecules:strandinvasion（鏈侵入）MovementoftheHollidayjunction:branchmigration（分支移動）CleavageoftheHollidayjunction:resolution（拆分）KeystepssharedbydifferentmodelsChapter10TheHollidaymodelverywellillustratestheDNAstrandinvasion,branchmigration,andHollidayjunctionresolutionprocessescentraltohomologousrecombination.TheHollidaymodelillustrateskeystepsinhomologousrecombination1.TheHollidaymodelHolliday模型中，同源重組的4個關(guān)鍵步驟①兩個同源染色體DNA排列整齊②一個DNA的一條鏈斷裂、并與另一個DNA對應(yīng)的鏈連接，形成Holliday中間體③通過分支移動產(chǎn)生異源雙鏈DNA④Holliday中間體切開并修復(fù)，形成兩個雙鏈重組體DNA，分別為：交換重組體(spliceorcrossoverrecombinant)補丁重組體(patchrecombinant)Chapter10Figure10-1Chapter10Resolution,finishingrecombination,requirescuttingoftheDNAThelocationofthecutsdetermineswhethertherecombinationresultsincrossoverproductsorpatchproductsHollidayModelFigure10-3Chapter10Cuttingthestrandsthatwere

notbrokenintheinitiationreactionresultsin“splice”orcrossoverproducts=regionsoftheparentalmoleculesarecovalentlylinkedbyregionofhybridduplexCuttingthestrandsthatwerebrokenininitiationresultsin“patch”product=aregionofhybridDNAinotherwiseparentalchromosomes,noreassortmentofflankinggenes=non-crossoverproductsHollidayModelCrossoverproductspatchproducts切開的鏈與原來斷裂的是同一條鏈，重組體含有一段異源雙鏈區(qū)，其兩側(cè)來自同一親本DNA。切開的鏈并非原來斷裂的鏈，重組體異源雙鏈區(qū)的兩側(cè)來自不同親本DNA。

補丁重組體(patchrecombinant)交換重組體(splicerecombinant)

ch10-1_hollidayChapter10Chapter10Homologousrecombinationisofteninitiatedbydouble-strandedbreaks(DSB)inDNA,whileHollidaymodelisinitiatebythepairofalignednicks.DSBoccursrelativelyfrequently,butthealignednicksnot.Thedouble-strandbreakrepairmodelmoreaccuratelydescribesmanyrecombinationevents2.Double-strandBreakRepairModelDSBsinDNAarisequitefrequently.Inbacteria,themajorbiologicalroleofhomologousrecombinationistorepairDSBs(Ionizingradiation,

damageintheDNAtemplate);DSBsalsopromotesgeneticexchange.Ineukaryoticcells,homologousrecombinationiscriticalforDNArepairing,aswellasfortheprocessofchromosomalpairingduringmeiosis.Double–strandedDNAbreaksarisebynumerousmeansandinitiatehomologousrecombinationChapter10Figure10-1.DamageintheDNAtemplatecanleadtoDSBformationduringreplication(復(fù)制叉瓦解)Chapter10Double-StrandedBreaksRecombinationisofteninitiatedbydsbreaksinDNA.InitiatingeventisaDSBin1of2molecules–morelikelymodelthanHollidaywhichrequires2ssbreaksatsameplacein2molecules.AfterinitiationDNA-cleavingmoleculesdegradebrokenDNAtoformssDNAtailsterminatingin3’OHends.DSBModelFigure10-4.DamageintheDNAtemplatecanleadtoDSBformationduringreplication.Chapter10

ssDNAtailsinvadetheunbrokenhomologousDNAduplex.Invasionmaybeinitiallybysinglestrand,then2invadingstrands,strandspairw/complementarystrand.Invadingstrandsserveas3’primersfornewDNAsynthesis.Elongationof3’endsusingthecomplementarystrandastemplatereplacesDNAdegradedbyprocessingatbreaksite.DSBModelFigure10-4.DamageintheDNAtemplatecanleadtoDSBformationduringreplication.Chapter10Chapter10

RegionofDNAreplacedbynewsynthesisis“l(fā)ost”.Geneconversion=replacementofregionononehomologousstrandw/sequencefromhomolog2Hollidayjunctionsmovebybranchmigration,extendingregionofexchangeasinHollidaymodel,andarethenresolvedbystandcleavage.DSBModelFigure10-4.DamageintheDNAtemplatecanleadtoDSBformationduringreplication.Chapter10

Cleavageofbothjunctionsatsite2willgiveapathproduct.Cleavageatbothjunctionatsite1alsogivesapatchproduct.Cleavageofonejunctionatsite1,buttheotheratsite2,thereforegeneratescrossoverproducts.Box1TwopossiblewaysofresolvinganintermediatefromtheDSB-repairpathway.Chapter10OUTLINEModelsforHomologousRecombination(twomodels)**HomologousRecombinationProteinMachines(RecBCDpathway)**HomologousRecombinationinEukaryotes(meiosisrecombination,proteins)Mating-TypeSwitching(ageneconversionevent)GeneticConsequencesoftheMechanismofHomologousRecombination(exchangeorconversion)Topic2:Homologousrecombinationproteinmachines---RecBCDpathwayChapter10Allorganismshaveenzymesthatcatalyzethebiochemicalstepsofrecombination.Somehomologousproteinfamiliesprovidethesamefunctioninallorganisms,otherrecombinationstepsarecatalyzedbydifferentclassesofproteinsindifferentorganisms.BestunderstoodmodelisfromE.coli,theRecBCDpathwayRecBCD具有多種酶活性，其主要的酶活性包括：ssDNA外切酶活性、dsDNA外切酶活性和解旋酶活性。解旋酶能夠在SSB存在情況下使雙鏈DNA解螺旋。RecBCD與dsDNA的末端結(jié)合，然后以大約每秒1000bp的速度解開DNA雙鏈，同時降解解旋產(chǎn)生的ssDNA。RecBCD對兩條單鏈的降解速度是不一致的，它優(yōu)先降解3`末端鏈。一個正在被RecBCD加工的DNA的末端會形成一個單鏈環(huán)和一個5`端拖尾。RecBCDDNAcomplexRecBCD的酶活性受重組熱點Chi的調(diào)節(jié)。Chi位點是大腸桿菌基因組中的一種不對稱的8bp核苷酸序列，5`-GCTGGTGG-3`，Chi位點能夠改變RecBCD的酶活性，它是大腸桿菌重組過程的必需組分，是重組的熱點。一旦RecBCD識別出Chi序列，RecBCD核酸酶活性便發(fā)生變化，其3’→5’外切酶活性受到抑制，5’→3’外切酶活性被激活，由原來優(yōu)先降解3’末端鏈，改變?yōu)橹唤到?’末端鏈。但是它的解旋酶活性未受到影響。PolaractionofchiChisitesincreasefrequencyofrecombinationatthesite10xChisiteisaspecificsequence(GCTGGTGG),butacommonsequence,infactitisoverrepresentedintheE.coligenome,1009insteadofthepredicted~80E.coliDNAcananddoincorporateDNAfromotherE.coliThisE.coliDNA,butnotviralorotherinvadingDNA,willhavechisites,DNAw/ochisiteswillbedegradedbyRecBCDFigure10-7Chapter10RecBCD是由3個亞基所組成復(fù)合體，具有多種酶活性，包括ssDNA外切酶活性、dsDNA外切酶活性和解旋酶活性。

RecBCD依靠ATP水解提供的能量，結(jié)合在DNA雙鏈斷裂處，并沿著DNA移動。

RecBCD的酶活性受重組熱點Chi的調(diào)節(jié)。NucleaseactivesiteFigure10-6.StructureoftheRecBCDDNAcomplex.RecBCDDNAcomplexChapter10RecBRecCRecDPriortochisite:在原核同源重組中存在8個堿基組成的Chi位點(5`-GCTGGTGG-3`);RecA、RecBCD參與重組。RecBCD復(fù)合體具有核酸酶，解旋酶和ATPase活性。在Chi位點產(chǎn)生單鏈3`游離未端.RecA是一種單鏈DNA結(jié)合蛋白，可催化一個雙鏈DNA分子的3`末端單鏈區(qū)侵入另一個雙鏈DNA分子，形成異源雙鏈區(qū)，同時置換出同源單鏈，形成Holliday結(jié)構(gòu)。RuvAB復(fù)合體特異性地識別Holliday結(jié)構(gòu)并促進異源雙鏈的形成（促進分枝移動）。RuvC是一種內(nèi)切酶，可以識別Holliday結(jié)構(gòu)并剪切DNA，從而終止重組。HomologousrecombinationinprokaryotesChapter10ProcessesbrokenDNAtogeneratesstails–thepreferredsubstrateforrecombination.loadsRecAproteinontossDNA.“selects”whetherDNAwillberecombinedordestroyed.RecBCDpathwayFigure10-5.StepsofDNAprocessingbyRecBCD.Chapter10RecBCDComplexFigure10-5.StepsofDNAprocessingbyRecBCD.RecBCDiscomposedof3subunits,productsoftherecB,recCandrecDgenesRecBCDentersDNAfromdsbreakandmovesalongtheDNARecBandRecDarebothhelicases,unwindingDNAusingenergyfromATPhydrolysisChapter10RecBCDnucleaseactivitydegradesDNAasitisunwoundAtchi(c)sequence,nucleaseactivityisaltered=RecBCDnolongercleavesthe3’to5’strand,andcleavestheotherstrandevenmoreThisasymmetricalcleavageresultsina3’tailterminatingatthechisequence–idealforassemblyofRecAandinitiationofrecombinationRecBCDpathwayFigure10-5.StepsofDNAprocessingbyRecBCD.Chapter10ChangeinnucleaseactivityappearstobeinactivationorlossofRecDsubunit3’overhangcouldalsobecoatedw/SSB,interactionofRecBCDandRecAensuresthatRecAbindsRecBCDpathwayFigure10-5.StepsofDNAprocessingbyRecBCD.Chapter10Figure10-8SubstratesforRecAstrandexchange.RecAproteinassemblesonsingle-strandDNAandpromotesstrandinvasionChapter10RecAbindstossDNAandisastrand-exchangeprotein,catalyzingthepairingofhomologousDNAmoleculesTheactiveformofRecAisaprotein-DNAfilament–huge,variablesize,100subunitsofRecA,300nucleotidesRecAfilamentcanbind1,2,3or4strandsCoatedwithRecAmakeDNAhighlyextended(0.34nm-0.5nm/bp)(延長1.5倍）PolarityofRecAassembly（5’-3’）Figure10-9ThreeviewoftheRecAfilament.RecAFilamentChapter10RecAbindingiscooperative,growingwiththeadditionofsubunitstothe3’sideofthefirstsubunit-inthe5’to3’direction=filamentendingina3’ismostlikelytobecoatedbyRecAPolarityofRecAassemblyFigure10-10Chapter10RecAbindstossDNARecA“l(fā)ooks”forsequencehomologyusing2bindingsites.ThessDNAisboundintheprimarybindingsite.dsDNAcanoccupythesecondarybindingsite–bindingisrapid,weak,transientandseq.independent.RecAmovesalongdsDNAscanningforhomology–base-flipping.ModeloftwostepsinsearchforhomologyandDNAstrandexchangewithintheRecAfilamentFigure10-11Chapter10Matchesof15+bptriggersstrandexchangeRecApromotesformationofstablecomplexbetweentwoDNAmolecules=three-strandedjointmoleculecontainingseveral100bpofhybridDNAThestrandintheprimarysiteispairedw/itscomplementintheduplexboundinthesecondarysite-breakingandforminghydrogenbondsFigure10-11Chapter10Strand-exchangeproteinsintheRecAfamilyarepresentinallorganisms.a)humanRad51,b)EcoliRecA,c)archaebacteriaRadA,allw/similarhelicalstructure.Figure10-12Chapter10TheRuvABcomplexrecognizesHollidayjunctionsandpromotesbranchmigration

Afterstrandinvasion,the2recombiningmoleculesareconnectedbyaHollidayjunction.Branchmigrationincreasestheamountofgeneticmaterialexchanged.RuvAisaHollidayjunctionDNA-bindingprotein,itbindsatjunctionandrecruitsRuvBtosite.RuvBisaATP-dependenthexamerichelicase,providesenergytodriveexchangeofbasesalongbranch.Figure10-13Chapter10HighresolutionstructureoftheRuvCresolvesandschematicmodeloftheRuvCdimerboundtoHollidayjunctionDNARuvCcleavesDNAat5’A/T-T-T-G/C3’sitesSequenceisspecific,butfrequent,~1/64nucleotidesModestsequencespecificityensuresacertainamountofbranchmigrationbeforeresolutionFigure10-14Chapter10

CompletionrequiresthatHollidayjunction(s)beresolved.RuvCisresolvingendonucleaseinbacteria,itfunctionsinconjunctionw/RuvABcomplex.RuvCrecognizesDNA-RuvABcomplexandnicks2DNAstrandsw/thesamepolarity,resulting5’and3’endsaresealedbyDNAligase.Dependingonwhichstrandsarecuttheproductsmay,ormaynot,beofthecrossovertype.Chapter10ch10_homologousChapter10OUTLINEModelsforHomologousRecombination(twomodels)**HomologousRecombinationProteinMachines(RecBCDpathway)**HomologousRecombinationinEukaryotes(meiosisrecombinationproteins)Mating-TypeSwitching(ageneconversionevent)GeneticConsequencesoftheMechanismofHomologousRecombination(exchangeorconversion)Topic3:Homologousrecombinationineukaryotes

Chapter10

發(fā)生在減數(shù)分裂過程中的同源重組有兩方面的重要作用。一是確保同源染色體能夠正確配對，而同源染色體的聯(lián)會是生殖細(xì)胞形成時染色體數(shù)目減半的基礎(chǔ)。另外，減數(shù)分裂重組也常常引起非姊妹染色單體之間的交換，結(jié)果是親本DNA分子上的等位基因在下一代發(fā)生了重新排列。Theduplicatedhomologouschromosomesmustpair.***Withoutrecombination,chromosomeoftenfailstoalignproperlyforthefirstmeioticdivision.Figure10-15DNAdynamicsduringmeiosisChapter10Figure10-16Meioticrecombinationbetweenhomologouschromatids.dsDNAchromatidsChapter10

在減數(shù)分裂的前期同源染色體開始配對的時候，SpoII蛋白在染色體的多個位置上切斷DNA。SpoII的切割位點多分布于染色體上核小體包裝疏松的區(qū)域。

ProgrammedgenerationofdoublestrandedDNAbreaksoccursduringmeiosisChapter10Figure10-18MechanismofcleavagebySpoII.Chapter10SpoII蛋白質(zhì)中的特異的酪氨酸側(cè)鏈可攻擊磷酸二酯骨架，切斷DNA，并形成蛋白質(zhì)-DNA共價復(fù)合物。SpoII的兩個亞基在兩個DNA鏈相差2個核苷酸的地方切開，形成一個交錯的雙鏈斷裂，機制類似拓?fù)涿?。切割后，?dǎo)致5`端的DNA共價連接到SpoII，產(chǎn)生鏈侵入。ManyproteinfunctiontogethertopromotemeioticrecombinationSomelargeprotein-DNAcomplexes,knownasrecombinationfactoriesRad51,Dmc1,Rad52……Chapter10MRXprotein：是由MreII、Rad50和Xre2三個亞基組成的DNA核酸酶。MRX酶作用于被切開的DNA末端，以組裝類RecA的鏈交換蛋白。Dmc1：是專一在減數(shù)分裂重組中行駛功能的類RecA蛋白。Rad51具有相似的功能。Chapter10

在真核細(xì)胞中已經(jīng)發(fā)現(xiàn)了兩種與細(xì)菌RecA蛋白同源的蛋白質(zhì)：Rad51和Dmc1。這兩種蛋白質(zhì)在減數(shù)分裂重組中發(fā)揮重要作用。Rad51在進行有絲分裂和減數(shù)分裂的細(xì)胞中廣泛表達(dá)，而Dmc1則僅在細(xì)胞進入減數(shù)分裂時被表達(dá)。依賴Dmc1的重組傾向于發(fā)生在非姊妹染色單體之間。減數(shù)分裂重組可能是通過促進同源染色體的交聯(lián)，而幫助待分離的同源染色體間的聯(lián)會的。首先，SpoII催化DNA雙鏈斷裂生成3`端單鏈區(qū)。在減數(shù)分裂重組中，MRX酶復(fù)合物負(fù)責(zé)催化5`到3`端的降解，并去除與DNA相連的SpoII。與細(xì)菌RecA同源的鏈交換蛋白質(zhì)Rad51和Dmc1結(jié)合在單鏈DNA尾上。MRXisnothomologoustoRecBCD,butisamultisubunitnucleaseDigestionsoccursonlyonthestrandw/5’endboundtoSpo11,5’to3’resection,generating+1kbofssDNAFigure10-19Meioticrecombinationpathway.Chapter10Eukaryoteshave2homologsofRecA:Rad51andDmc1Rad51isexpressedincellsundergoingmitosisormeiosis,Dmc1isexpressedonlyduringmeiosisTogetherRad51andDmc1participateinrecombination,buthowtheyinteractisnotknownFigure10-19Meioticrecombinationpathway.Chapter10Manyproteinsinteractinhomologousrecombination,likelyasalargemulticomponentcomplexesTheserecombinationfactoriescanbevisualizedinthecell,e.g.co-localizationofRad51andDmc1duringmeiosisChapter10Rad52interactsw/Rad51topromoteformationofRad51-DNAcomplexes,antagonizesactionofRPA,thenormalssDNAbindingprotein.Mus81isrequiredformeiosisandmayfunctiontopromotebranchmigrationandHollidayjunctionresolution.OtherrecombinationproteinsineukaryotesChapter10Chapter10OUTLINEModelsforHomologousRecombination(twomodels)**HomologousRecombinationProteinMachines(RecBCDpathway)**HomologousRecombinationinEukaryotes(meiosisrecombination,proteins)Mating-TypeSwitching(ageneconversionevent)GeneticConsequencesoftheMechanismofHomologousRecombination(excha