分子生物學(xué)課件第五章-同源重組、位點(diǎn)特異性重組

第五章同源重組、位點(diǎn)特異性重組及轉(zhuǎn)座作用鄭偉娟2006DNARecombinationRolesTypesMechanismDNA重組（recombination）---是指發(fā)生在DNA分子內(nèi)或DNA分子之間核苷酸序列的交換、重排和轉(zhuǎn)移現(xiàn)象，是已有遺傳物質(zhì)的重新組合過程。RolesGeneratingnewgene/allelecombinations (crossingoverduringmeiosis)Generatingnewgenes(e.g.,IgGrearrangement)IntegrationofaspecificDNAelementDNArepair

PracticalUsesUsedtomapgenesonchromosomes (recombinationfrequencyproportionaltodistancebetweengenes)MakingtransgeniccellsandorganismsTypesHomologous-occursbetweensequencesthatarenearlyidentical(e.g.,duringmeiosis)Site-Specific-occursbetweensequenceswithalimitedstretchofsimilarity;involvesspecificsitesTransposition–DNAelementmovesfromonesitetoanother,usuallylittlesequencesimilarityinvolved發(fā)生在DNA的同源序列之間；同源重組包括細(xì)菌的接合（conjugation）、轉(zhuǎn)化（transformation）和轉(zhuǎn)導(dǎo)（transduction）以及真核細(xì)胞的在同源染色體之間發(fā)生的交換等；發(fā)生在DNA的特異位點(diǎn)之間；噬菌體DNA與宿主菌染色體DNA的整合，如λ噬菌體與大腸桿菌的位點(diǎn)特異性整合；位點(diǎn)特異性重組不依賴于序列的同源性；轉(zhuǎn)座作用與轉(zhuǎn)座子（Transposon）的復(fù)制有關(guān);7.1同源重組HomologousRecombinationExamplesofhomologousRecombinationFig.22.1TheHollidayModelThismodelofrecombinationwasfirstproposedbyRobinHollidayin1964andre-establishedbyDavidDresslerandHuntingtonPotterin1976whodemonstratedthattheproposedphysicalintermediatesexisted.AligntwohomologousDNAmoleculesNicktheDNAatthesameplaceonthetwomolecules

ExchangestrandsandligateTheintermediatethatisformediscalledaHollidayintermediateorHollidaystructure.

onemoleculeisnowrotatedthrough180owithrespecttotheotherResolvethestructure

Thebasic(simple)model

NoncrossoverrecombinationCrossoverrecombinationAmorerealisticmodel

Branchmigration.patchspliceNoncrossoverrecombinationCrossoverrecombinationFirstproposedbyMatthewMeselsonandCharlesRaddingin1975.Double-strandbreakinexactlycorrespondingpositionsisnotrequired.TheMeselson-RaddingModelFig.22.2patchNoncrossoverrecombinationCrossoverrecombinationMeselson

Radding

DSBRepairModelDSBisinitiatingeventCutduplexisdonorofgeneticinformationEnzymesarenotsequencespecificFirststableintermediatebetweentwochromatidsisathreestrandedstructureTheRecBCDPathwayActuallyrecombinationpathwayusedbyE.coli.AvariationofMeselson-Raddingmodel.BeginwithRecBCDproteincreatinganicknearthe3’endofchisite,χ,5’-GCTGGTGG-3’.Chisitesarefoundonavarageevery5000bpintheE.coligenome.RecBCDmultifunctionprotein:DNAhelicaseactivityssDNAanddsDNA

exonucleaseactivityssDNA

endonucleaseactivity1.RecBCDproteinnicksonestrandtothe3’-sideofthechisite.2.TheDNAhelicaseactivityofRecBCDthenunwindstheDNAthroughthechi-siteformingasingle-strandtail.3.

RecA

andSSBcoatthetail.4.RecApromotesinvasionofanotherDNAduplex,formingaD-loop.5.RecAhelpstheinvadingstrandscanforaregionofhomologyintherecipientDNAduplex.6.Theinvadingstrandbase-pairedwithahomologousregion,releasingSSBandRecA.7.RecBCDnicksthelooping-outstrand.RecAandSSBhelpsstrandexchange.Fig.22.5f-h8.ThenicksaresealedbyDNAligase,yieldingaHollidayjunction.9.Branchmigrationoccours,sponsoredbyRuvAandRuvB.10.RuvCresolvesthestructure.TheMainProteinsinvolved

inhomologousrecombinationinE.

coli

RecARecBCD

RuvARuvB

RuvC

RecA

TheRecAproteinisamultifunctionalpowerhouse!Ithasstrand-exchange,ATPaseandco-proteaseactivitiesallpackedintoacompact352amino-acid,38kDastructure.ItisrequiredforallrecombinationpathwaysinE.

coli.TheRecAproteinwillbindcooperativelytoassDNAmoleculewitheachmonomerofRecAbindingtoaspanof4-6nucleotides.Assemblyofthenucleoproteincomplexproceedsina5‘→3’direction.Thecomplexisfairlystable(half-lifeis30min)andistheactivespeciesthatwillpromotestrandexchange.RecAwillpromotestrandexchangebetweenDNAmoleculesaslongasthefollowingconditionsapplyOneofthetwomoleculesmusthaveassDNAregiontowhichRecAcanbind.

Thetwomoleculesmustsharearegionofhomologous(i.e.nearlyidentical)DNAsequence-aminimumof30-151bpisrequired.

Theremustbeafreeendwithinthisregionofhomologywhichcaninitiatethestrandexchange.

ThreestagesofparticipationofRecAinstrandexchangePresynapsis:

RecAandSSBcoatthessDNA;Synapsis:alignmentofcomplementarysequencesinthessDNAanddsDNA;Postsynapsis:strandexchange;*RecAbindspreferentiallytothesingle-strandDNA,andSSBacceleratesthisprocedurebyunwindsecondarystructureofthesingle-strandDNAparticipatinginrecombination.*SynapsiscanoccurefficientlywhenassDNAfindahomologousregion(atleast30-151bp)inadsDNAwiththepresenceofRecA.ThetwoalignedDNAstrandsaresidebyside,butnotinterwinded.*RecAandATPcollaboratetopromotestrandexchange.RecAhasATPaseactivity,whichhydrolysisATPtodissociateRecAfromsynapsisDNAandmakewayforstrandexchange.RecBCD

TherecB,recC&recDgenescodeforthethreesubunitsoftheRecBCDenzyme.RecBCDisalsoaversatileenzymewith:i)aDNA-dependentATPaseactivity;ii)aDNAnucleaseactivity;actsonbothssDNAanddsDNA,cleavesspecificallyatchisite.

iii)adsDNA

helicaseactivity;1.RecBCDproteinnicksonestrandtothe3’-sideofthechisite.2.TheDNAhelicaseactivityofRecBCDthenunwindstheDNAthroughthechi-siteformingasingle-strandtail.3.

RecAandSSBcoatthetail.4.RecApromotesinvasionofanotherDNAduplex,formingaD-loop.5.RecAhelpstheinvadingstrandscanforaregionofhomologyintherecipientDNAduplex.6.Theinvadingstrandbase-pairedwithahomologousregion,releasingSSBandRecA.7.RecBCDnicksthelooping-outstrand.RecAandSSBhelpsstrandexchange.*TheRecBCDcomplexcanfunctionasaDNA

exonuclease.Itwillbindtodouble-strandedbreaksinDNAanddegradebothstrandssimultaneously.*TheRecBCDcomplexalsohasendonucleaseactivity.

ItcannicknearthechisitewhenitencountersaChisequence.

ThentheRecDsubunitisreleasedandtheRecBCproteinsactasahelicasetounwindtheDNAinanATPdependentreaction.ThisgeneratesassDNAregionthatcanserve(alongwithRecA)toinitiatestrandexchangeandarecombinationreaction.TheRecBCD

helicaseactivitycanunwindDNAfasterthanitrewinds.ThusasittravelsalongaDNAmolecule,itcangeneratessDNAloops.RuvAandRuvBProteinsneededinbranchmigration;FormaDNAhelicasethatcatalyzesthebranchmigration;RuvAtetramerbindstoHJ(eachDNAhelixbetweensubunits);

RuvBisahexamerring,hashelicase&ATPaseactivity;2copiesofruvBbindattheHJ(toruvAand2oftheDNAhelices);*RuvBcandrivebranchmigrationbyitselfinhighconcentration,soithastheDNAhelicaseactivityandATPaseactivity.*RuvAholdstheHJinasquareplanarconformation,facilitatesbindingofRuvBandthuspromotebranchmigration.ruvBruvADirectionofbranchmigration,awayfromRuvB

RuvC:resolvaseBindstoHJasadimer

Endonucleasethatcuts2strandsofHJConsensussequence:(A/T)TT(G/C)-occursfrequentlyinE.coligenome -branchmigrationneededtoreachconsensussequence!Fig.22.31aRuvA,RuvBandRuvCworktogetherintheformofRuvABC-junctioncomplex,or“resolvasome”.RuvBcanbindtoRuvA,andRuvCcanbindtoRuvB.MeioticRecombinationTookplaceinmosteukaryotes;Sharemanycharacteristicsincommonwithhomologousrecombinationinbacteria;Theinitiatingeventisquitedifferentfromthatofbacteria;7.2位點(diǎn)特異性重組Site-specificRecombinationSite-specificrecombination

Asthenameimplies,thistypeofrecombinationinvolvestheexchangeofgeneticmaterialatveryspecificsitesonly.RequireslesssequencehomologybetweenrecombiningDNAsthandoeshomologousrecombination.Specializedproteinsareneededtorecognizethesesitesandtocatalyzetherecombinationreactionatthesesites.Thestepsandfeatures

StrandexchangeFormationofahollidayintermediateBranchmigrationResolutionbetweentwoDNAmolecularswithinoneDNAmolecularInvertedrepeatsDirectrepeatsExamplesofSite-SpecificRecombination

IntegrationofbacteriophagelambdaFlagellarantigenvariationinSalmonellatyphimurium

DiversityofIgGgenesSite-specificintegrationofl

attPbindsInt,IHFcomplexbindsattB

Intrecombinesthetwomoleculesusingthematch“O”sequence

Xisremoves“l(fā)ysogenic”phageinresponsetoenvironmentalstressMechanismofIntaction

ATPindependentprocess5’OHand3’phosphatesCovalentTyrattachment-akintotopoisomerases,Spo11FlagellarPhasevariation

AlsoinphageMu,P1andP7.7.3轉(zhuǎn)座作用DNAtranspositionDNAtransposition

BarbaraMcClintockspentmanyyearspatientlystudyingthebehaviorofunusualgeneticelementsinmaize.Sheconcludedthattheseelementswere,infact,mobile.Herwork,allthemoreamazingbecausemuchofitwascarriedoutbeforethestructureofDNAwassolved,waslargelyignoreduntilthemid1970swhensimilarelementswerediscoveredinbacteria.TransposableGenetic

ElementsinBacteria

InsertionSequencesSimpletransposonsCompositeTransposons

BacteriophageElements(Mu)InsertionSequencesInsertionSequencesorISelementsarethefirsttransposonsdiscoveredinbacteria.thesimplestmobileelementconsistofafairlyshort(700-1500bp)DNAsegmentflankedbya10-40bpinvertedrepeatsequence.Thesegmentcodesfortheprotein(transposase)thatcatalysesthetranspositionevent:*轉(zhuǎn)座發(fā)生后在轉(zhuǎn)座子兩側(cè)產(chǎn)生一對(duì)短的直接重復(fù)序列SimpletransposonssimilartoISelements.containDNAsegmentsflankedbyshortinvertedrepeatsequences.TheDNAsegments,however,usuallycodeforanumberofgeneproducts：transposase,resolvase

，antibioticresistancegenes:CompositeTransposons

FlankedbyanISelementateitherend.EachISelementisatypicalISelementalthoughonlyoneofthetwoelementstypicallyretainsafunctionaltransposaseactivity.TheISelementsmaybeinthesameorintheoppositeorientationwithrespecttooneanother.TheinterveningsegmentoftencarriesthegeneticdeterminantsforanumberofantibioticorothertoxinresistancesBacterio