分子生物学教学课件：第11章 Site-Specific Recombination Transposition of DNA

第9章：1.有哪些因素会导致DNA突变和损伤？2.DNA突变和损伤其修复机制,每种修复机制中设计涉及的关键蛋白或酶有哪些，他们的主要作用是什么。第10章:1.同源重组的两个主要的模型是什么？2.同源重组过程中的关键步骤有哪些？3.大肠杆菌中在同源重组过程中涉及的关键蛋白有哪些，它们的主要作用是什么？Chapter11上一节内容回顾Site-SpecificRecombination&TranspositionofDNAChapter11AlthoughDNAreplication,repair,homologousrecombinationoccurwithhighfidelitytoensurethegenomeidentitybetweengenerations,therearegeneticprocessesthatrearrangeDNAsequencesandthusleadtoamoredynamicgenomestructure.Chapter11GenomeStable:Dynamic:DNAreplicationrepairhomologousrecombinationConservativesite-specificrecombination(CSSR)Transpositionalrecombination(Transposition)

ImpactonthechromosomestructureandfunctionKeymechanism:RecombinaseRecognizeGatherCleavageRejoinInverseormoveChapter11aTwoclassesofgeneticrecombinationforDNArearrangement:Conservativesite-specificrecombination(CSSR):recombinationbetweentwodefinedsequenceelementsTranspositionalrecombination(Transposition):recombinationbetweenspecificsequencesandnonspecificDNAsitesChapter11Figure11-1Conservativesite-specificrecombinationTranspositionalrecombinationChapter11OUTLINE1.ConservativeSite-SpecificRecombination.2.BiologicalRolesofSite-SpecificRecombination3.Transposition4.ExamplesofTransposableElementsandTheirRegulation5.V(D)JRecombinationChapter11Topic1ConservativeSite-SpecificRecombination(CSSR)CSSRisrecombinationbetweentwodefinedsequenceelementsExchangeofnon-homologoussequencesatspecificDNAsites(what)

MediatedbyproteinsthatrecognizespecificDNAsequences.(how)Chapter111-1CSSRoccursatspecificDNAsequencesinthetargetDNACSSR(conservedsite-specificrecombination)isresponsibleformanyreactionsinwhichadefinedsegmentofDNAisrearranged.Chapter11ConservativeSite-SpecificRecombinationspecificDNAsitesinthetargetDNASite-SpecificRecombinaseFigure11-2Integrationofλgenomeintothechromosomeofthehostcell.Chapter11CSSRcangeneratethreedifferenttypesofDNArearrangementsFigure11-3Chapter11RecombinationsiteStructuresinvolvedinCSSR:RecognitionsequencesCrossoverregion:

asymmetricPolarityInvertedrepeatDirectrepeatFigure11-4StructuresinvolvedinCSSRChapter111-2Site-specificrecombinasescleaveandrejoinDNAusingacovalentprotein-DNAintermediateThere’retwofamiliesofconservativesite-specificrecombinases:1.SerineRecombinases2.TyrosineRecombinasesChapter11Figure11-5Covalent-intermediatemechanismusedbytheserineandtyrosinerecombinasesChapter11蛋白质-DNA共价中间体中，蛋白质和DNA之间的共价连接保留了断裂的磷酸二酯键的能量。这样DNA链可以通过逆转断裂过程被重新连接。这种机制的特性提供了保守性位点特异性重组，被称为“保守”是因为反应中被断开的每个DNA键都被重组酶重新连接起来，而DNA在被重组酶断开并重新连接的过程中不需要其他的能量。这种机制也常见于DNA拓扑异构酶和Spo11，后者运用这种机制在减数分裂期间诱导DNA双链断裂引发同源重组。Chapter111-3SerinerecombinasesintroduceDSBinDNAandthenswapstrandstopromoterecombinationFirst,theserinerecombinasescleaveallfourstrandsSecond,DNAswapoccursFinally,theserinerecombinasesareliberatedandtheysealtheDNAstrandsChapter11RecombinationbyaserinerecombinaseFigure11-6Chapter111-4TyrosinerecombinasesbreakandrejoinonepairofDNAstrandsatatimeIncontrasttotheserinerecombinases,thetyrosinerecombinasescleaveandrejointwoDNAstrandsfirst,andonlythencleaveandrejointheothertwostrands.Chapter11RecombinationbyatyrosinerecombinaseFigure11-7Chapter111-5

StructureoftyrosinerecombinasesboundtoDNArevealthemechanismofDNAexchangeCreisatyrosinerecombinase.CreisanphageP1-encodedprotein,functioningtocircularizethelinearphagegenomeduringinfection.TherecombinationsitesofCreisloxsites.Cre-lox

issufficientforrecombination.Chapter11Figure11-9Mechanismofsite-specificrecombinationbytheCrerecombinase.Chapter11Topic2Biologicalrolesofsite-specificrecombinationChapter11ManyphageinserttheirDNAintothehostchromosomeduringinfectionusingthisrecombinationmechanism.Example:lphageAltergeneexpression.Example:SalmonellaHinrecombinaseMaintainthestructuralintegrityofcircularDNAmoleculesduringcyclesofDNAreplication.Example:resolvasethatresolvesdimertomonomer.Chapter11AllreactionsdependcriticallyontheassemblyoftherecombinaseproteinontheDNAandbringtogetherofthetworecombinationsites.Somerecombinationrequiresonlytherecombinaseanditsrecognitionsequenceforsuchanassembly;somerequiresaccessoryproteinsincludingArchitecturalProteinsthatbindspecificDNAsequencesandbendtheDNA.Thegeneralthemesofsite-specificrecombinationChapter11

2-1

lintegrasepromotestheintegrationandExcisionofaViralGenomeintotheHostCellChromosomeBacteriophagelinfectsahostbacteriumandwouldestablishalysogen,whichrequirestheintegrationofphageDNAintohostchromosomeTointegrate,lIntcatalyzesrecombinationbetweentwospecificsites—attachment(att)sites

attPsiteisonthephageDNAand

attBsiteisonthebacterialgenome

lIntisatyrosinerecombinase,andthemechanismofstrandexchangefollowsthepathwaydescribedabovefortheCreproteinChapter11lgenomeintegration.Recombinationalwaysoccursatexactlythesamesequencewithintworecombinationsites,oneonthephageDNA,andtheotheronthebacterialDNA.Chapter11Int

(l-encodedintegrase)Xis(l-encodedexcisionase)IHF(integrationhostfactorencodedbybacteria)Figure11-10Chapter11tyrosinerecombinaseIntbindingsite

C---Intbindingsite

H---IHFbindingsite

X---Xisbindingsite

P---IntbindingsiteCrossoverregionsPhagegenomeBacterialgenomeIHFbindingtotheH`sitebendstheDNAtoallowonemolecueoflInt

tobindboththeP1`andC1`site.Figure11-11Chapter11

PhagelexcisionrequiresanewDNA-bindingproteinPhagelexcisionrequiresanarchitecturalproteincalledXis,whichisphage-encoded.XisbindstotheintegratedattRsitestostimulateexcisionandtoinhibitintegration.Chapter112-2

TheHinrecombinase

invertsasegmentofDNAallowingexpressionofalternativegenes

TheSalmonellaHinrecombinaseinvertsasegmentofthebacterialchromosometoallowexpressionoftwoalternativesetsofgenes.HinrecombinaseisanexampleofprogrammedrearrangementsinbacteriaInthecaseofHininversion,recombinationisusedtohelpthebacteriaevadethehostimmunesystemHinisaserinerecombinasewhichpromotesinversion

Chapter11fljBencodesH2flagellinfljAencodesatranscriptionalrepressorofH1flagellinFigure11-13DNAinversionbytheHinrecombinaseofSalmonella.Chapter11HinrecombinationrequiresaDNAenhancerHinrecombinationrequiresaDNAenhancerinadditiontothehixsites.EnhancerfunctionrequiresthebacterialFisprotein.theenhancer-Fiscomplexactivatesthecatalyticstepsofrecombination.Hin-catalyzedinversionisnothighlyregulated,rather,inversionoccursstochastically.Chapter11AFis:factorforinversionstimulation,contactwithHintoformtheinvertasome（倒位体）ENSURETHATrecombinationoccursonsameDNAmolecule.Figure11-14Chapter112-3

Recombinase

convertsmultimericcircularDNAmoleculesintomonomersCircularDNAmoleculessometimesformdimersandevenhighermultimericformsduringtheprocessofhomologousrecombination.

Site-specificrecombinases(sometimescalledresolvases)canresolvedimersandlargermultimersintomonomers.Chapter11Figure11-15CircularDNAmoleculescanformmultimersChapter11

Xerrecombinaseisatyrosine.Xercatalyzesthemonomerizationofbacterialchromosomesandofmanybacterialplasmids.Xerisaheterotetramer,containingtwosubunitsofXerCandtwosubunitsofXerD.XerCandXerDrecognizedifferentsequence.ThedirectionalregulationofXer-mediatedrecombinationisachievedthroughtheinteractionbetweentheXerrecombinaseandacelldiversionproteincalledFtsK.Chapter11XerrecombinaseBox11-2Fig1ThedimeronlyresolveswhenXerDisactivatedbythepresenceofFtsKChapter11Topic3Transposition(转座)Transposition

isaspecificformofgeneticrecombinationthatmovescertaingeneticelementsfromoneDNAsitetoanother.Thesemobilegeneticelementsarecalledtransposableelements

or

transposons.MovementoccursthroughrecombinationbetweentheDNAsequencesattheendsofthetransposonsandasequenceinthehostDNAwithlittlesequenceselectivity.Chapter11

3-1SomegeneticelementsmovetonewchromosomallocationsbytranspositionFigure11-16TranspositionofamobilegeneticelementtoanewsiteinhostDNAChapter11Thetransposonscaninsertwithingenesorregulatorysequenceofagene,whichresultsinthecompletelydisruptionofgenefunction.Theycanalsoinsertwithintheregulatorysequencesofagenewheretheirpresencemayleadtochangesinhowthatgeneisexpressed.Transposableelements

arepresentinthegenomesofalllife-forms.(1)transposon-relatedsequencescanmakeuphugefractionsofthegenomeofanorganism.(2)thetransposoncontentindifferentgenomesishighlyvariable.Chapter11Transpositioningenome:occurrenceanddistributionGreen:mostlycomposedoftransposon-relatedsequences.Purple:cellulargenes.Figure11-17

Chapter113-2

TherearethreeprincipleclassesoftransposableelementsChapter11怎么来的Chapter113-2-1DNAtransposonscarryatransposasegene,flankedbyrecombinationsitesRecombinationsitesareatthetwoendsofthetransposonandareinvertedrepeatedsequencesvaryinginlengthfrom25toafewhundredbp.Therecombinaseresponsiblefortranspositionareusuallycalledtransposasesorintegrases.Sometimestheycarryafewadditionalgenes.Example,manybacterialDNAtransposonscarryantibioticresistancegene.Chapter11TransposonsexistasbothautonomousandnonautonomouselementsAutonomoustransposons:carryapairofterminalinvertedrepeatsandatransposasegene;functionindependentlyNonautonomoustransposons:carrytheterminalinvertedrepeatsbutnotthefunctionaltransposase;needthetransposaseencodedbyautonomoustransposonstoenabletransposition3-2-2Viral-likeretrotransposonsandretrovirusescarryterminalrepeatsequencesandtwogenesimportantforrecombinationInvertedterminalrepeatsequencesforrecombinasebindingareembeddedwithinlongterminalrepeats(LTRs),beingorganizedonthetwoendsoftheelementsasdirectrepeats.Reversetranscriptase(RT;反转录酶),usinganRNAtemplatetosynthesizeDNA.Integrase

(整合酶)orthetransposase(转座酶)Chapter113-2-3

Poly-AretrotransposonslooklikegenesDonothavetheinvertedterminalrepeats.Onendiscalled5’UTR(untranslatedregion),theotherendis3’UTRfollowedbyastretchofA-Tbasepairscalledthepoly-Asequence.Flankedbyshorttargetsiteduplication.Carrytwogenes.ORF1encodesanRNA-bindingproteins.ORF2encodesaproteinwithbothreversetranscriptase(RT)andendonucleaseactivity.Truncatedelementslackingcomplete5’UTR??Chapter113-3DNAtranspositionbyacut-and-pastemechanism

ThemovementofaDNAtransposonbyanon-replicativemechanismcalledcut-and-pastetransposition.1.First,transposasebindstotheinvertedterminalrepeatsattheendofthetransposonandbringsthetwoendsofthetransposonDNAtogethertogenerateastableprotein-DNAcomplexcalledthesynapticcomplex

or

transpososome.Chapter112.Next,thetransposonDNAisexcisedfromitsoriginallocationinthegenome.3.Then,the3’-OHendsofthetransoponDNAattacktheDNAphosphodiesterbondsatthesiteofthenewinsertion,thisDNAsegmentiscalledthetargetDNA.4.Atlast,thetransposonDNAiscovalentlyjoinedtotheDNAatthetargetsitebyDNAstrandtranfer.ThisreactionintroducedanickintothetargetDNA.Chapter11Chapter11Figure11-19Thecut-and-pastemechanismoftransposition.转座酶断裂DNA链的位置在转座子DNA和它所插入的宿主序列间的连接处，叫flankinghostDNA。1.TwointroducednickarefilledbyaDNArepairpolymerase(encodedbythehostcell)andaDNAligase2.FillinginthegapgivesrisetothetargetsiteduplicationsthatflanktransposonsTheintermediateincut-and-pastetranspositionisfinishedbygaprepairChapter111.Anenzymeotherthantransposasecanbeusedtocleavethenontransferedstrand2.ThetransposaseitselfcleavethenontransferedstrandbyusinganunusualDNAtransesterification(转酯基)mechanism3.DNAcleavageviaatransesterificationreactioncanalsooccurbetweentwoendsofthetransposon3-4TherearemultiplemechanismsforcleavingthenontransferredstrandduringDNAtranspositionChapter11FIGURE12-21Threemechanismsforcleavingthenontransferredstrand.3-5DNAtranspositionbyareplicativemechanismFirst,thetransposaseproteinisassembledonthetwoendsofthetransposonDNAtogenerateatranspososome.Then,DNAiscleavedattheendsofthetransposonDNA.Figure11-22Chapter11

Then,the3’OHendsofthetrsnsposonDNAarejoinedtothetargetsitesbytheDNAstrandtransferreaction,whichgenerateadoublybranchedDNAmoleculeAtlast,ThetwoDNAbrancheswithinthisintermediatehavethestructureofareplicationfork,andtheDNAsynthesisisproceeded

ThisreplicationreactiongeneratestwocopiesofthetransposonDNAFigure11-22Chapter113-6

Viral-likeretrotransposonsandretrovirusesmoveusinganRNAintermediateRecombinationforretroelementsinvolvesanRNAintermediate.Acycleoftranspositionstartswithtranscriptionoftheretrotransposon(orretroviral)DNAsequenceintoRNAbycellularRNApolymerase.TranscriptioninitiatesatapromotersequencewithinoneoftheLTRs.

TheRNAisthenreversetranscribedtogeneratethecDNAChapter113.

ThecDNAisrecognizedbyIntegraseandrecombinatewithanewtargetDNAsite4.IntegraseassemblesontheendsofthiscDNAandcleavesafewnucleotidesoffthe3’endsofeachstrand5.Integrasecatalyzestheinsertionofcleaved3’endsintoaDNAtargetsiteinthehostcellgenomeusingtheDNAstrandtransferreaction.6.Gaprepairreactiongeneratestarget-siteduplications.Chapter11Figure11-23Chapter11Mechanismofretroviralintegrationandtranspositionofviral-likeretrotransposons.RNA没有携带全长的LTR序列，在逆转录过程中，有两个内部引物合成和两个单链转换反应（见框11-3）。3-7DNAtransposasesandretroviralintegrasesaremembersofaproteinsuperfamilyManydifferenttranposasesandintegrasescarryacatalyticdomainthathasacommonthree-dimensionalshapeThisdomaincontainstwoAsp(D)andaGlu(E)

Thetranposase/integraseproteinsusethissame

sitetocatalyzeboththeDNAcleavageandtheDNAstrandtransferTranposasesandintegrasesareonlyactivewhenassembledintoasynapticcomplex,alsocalledatranspososome,onDNAChapter11Tn5tranposaseMutranposaseRSVintegraseFIGURE12-24Similaritiesofcatalyticdomainsoftransposasesandintegrases.3-8

Poly-ARetrotranspositionmovebya“reversesplicing”mechanismThePoly-ARetrotransposonsuseanRNAintermediatebutuseamechanismdifferentthanthatusedbytheviral-likeelements.Thismechanismiscalledtargetsiteprimedreversetranscription1.First,theDNAofanintegratedelementistranscriptedbyacellularRNApolymerase2.Then,newlysynthesizedRNAisexportedtocytoplasmtoproduceORF1andORF2proteinsChapter113.

Theprotein-RNAcomplexthenreentersthenucleaseandassociateswiththecellularDNA.(TheORF2proteinhasbothaDNAendonucleaseactivityandareversetranscriptaseactivity.)4.TheendonucleaseinitiatestheintergrationreactionbyintroducinganickinthechromosomalDNA(ployT).5.The3’OHDNAendgeneratedbythenickingactionthenservesastheprimerforreversetranscriptionoftheelementRNA.Chapter11Figure11-26Chapter11Transpositionofapoly-Aretrotransposonbytargetsite-primedreversetranscriptionFigure11-26Topic4ExamplesoftransposableenementsandtheirregulationTwotypesofregulationappearasrecurringthemes:Transposonscontrolthenumberoftheircopiespresentinagivencell.Trnasposonscontroltargetsitechoice.Chapter114-1

IS4-familytransposonsarecompactelementswithmultiplemechanismsforcopynumbercontrolTn10transposesviathecut-andpastemechanism,usingtheDNAhairpinstrategytocleavethenontransferedstrands.Tn10limitsitscopynumberinanygivencellbystrategiesthatrestrictitstranspositionfrequency.OnemechanismistheuseofanantisenseRNAtocontroltheexpressionofthetransposasegene.Bythismechanism,cellsthatcarrymorecopesofTn10willtranscribemoreoftheantisenseRNA,whichinturnwilllimitexpressionofthetransposasegene.Thetranspositionfrequencywill,therefore,beverylowinsuchastrain.Chapter11Tn10Structure:Mechanism:cut-and-pastemechanismChapter11Anti-senseRNAAntisenseregulationofTn10expressionFigure11-28Chapter114-2Tn10transpositioniscoupledtocellularDNAreplicationBacteriamethylatetheirDNAatGATCsitesandGATCsitesarehemimethylatedforafewminutes.Itisduringthebriefperiod—whentheTn10DNAishemimethylated—thattranspositionismorelikelytooccur.BothRNApolymeraseandtransposasebindmoretightlytothehemimethylatedsequencesthantotheirfullymethylatedversions.Asaresult,whentheDNAishemimethylated,thetransposasegeneismostefficientlyexpressed,andthetransposaseproteinbindsmostefficientlytotheDNA.Chapter11TranspositionofTn10afterpassageofareplicationfork.Figure11-29Chapter114-3PhageMuisanextremelyrobusttransposonRobusttransposonTranspositionwayissimilartoretrovirusesCarrytwogenesrelatedwithtranspotion:MuAMuBRegulation:targetimmunitytoavoidtransposingintoitsownDNA15kbChapter11Figure11-30EarlystepsofMutranspositionChapter11Topic5

V(D)JrecombinationTheprincipalmechanismcellsusetogenerateantibodiesandTcellreceptorswithsuchdiversityreliesonaspecializedsetofDNArearrangementreactionsknownasV(D)JrecombinationChapter11OverviewoftheprocessofV(D)JrecombinationChapter11Figure11-35TheearlyeventsinV(D)JrecombinationoccurbyamechanismsimilartotransposonexcisionRecombinationsequences,calledrecombinationsignalsequences,flankthegenesegmentsthatareassembledbyV(D)Jrecombination.RecombinationalwaysoccursbetweenapairofrecombinationsignalsequenceswhichareorganizedasinvertedrepeatsflankingtheDNAsegmentsthataredestinedtobejoined.Chapter11Fig11-36RecombinationsignalsequencesrecognizedinV(D)Jrecombination.First,reconbinaserecognizestherecombinationsignalsequencesandpairsthetwositestoformaprotein-DNAsynapticcomplex.Then,theRAG1proteinswithinthiscomplexintroducesingle-strandedbreaksintheDNAateachofthejunctionsbetweentherecombinationsignalsequenceandthegenesegmentthatwillberearranged.Then,this3’OHDNAendattackstheoppositestrandoftheDNAdoublehelix,whichresultsinthegenerationofahairpinDNAend.Chapter11TheV(D)JrecombinationpathwayChapter11Figure11-37KeyPoint1.概念：位点特异性重组(保守性位点特异性重组)、DNA转座、转座子、V(D)J重组、自主转座子和非自主转座子等。2.丝氨酸/络氨酸重组酶的重组机制3.DNA转座的特点。4.三种主要转座子的结构特点。5.DNA转座的剪切-粘贴机制和复制机制。Completelyunderstandtwoanimationsaboutsite-specificrecombinationandtranspositionofDNA.本章要点

1.名词：位点特异性重组(保守性位点特异性重组)、DNA转座、转座子、V(D)J重组、自主转座子和非自主转座子等。2.