11CCSR and Transposition转座子.docx

11CCSR and Transposition转座子.docx

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11CCSRandTransposition转座子

Site-SpecificRecombinationandTranspositionofDNA

Chapter11

A.Conservativesite-specificrecombination（CSSR）

B.Transposition

C.TransposableElementsinEukaryotes

Thereare2maintypesofgeneticrecombination:

conservativesite-specificrecombination（CSSR）

transpositionalrecombination（usuallyjustcalledtransposition）

CSSRisrecombinationbetween2specificsequences.TherearerecombinationsiteswhereDNAexchangeoccurs.

Transpositionisrecombinationbetweenspecificsitesonatransposableelementandnonspecificsitesatthesiteofinsertion.

CSSRandtranspositionhavesimilarmechanisms.Bothhaverecombinasesthatrecognizespecificsequenceswhererecombinationwilloccur.

TherecombinasesbindtheseDNAsequencesandcatalyzecuttingtheDNAandthenrejoiningit.

A.ConservativeSite-SpecificRecombination

Conservativesite-specificrecombinationdiffersfromtranspositioninthatshortspecificsequences,whicharerequiredfortherecombination,aretheonlysitesatwhichrecombinationoccurs.Thesesequencesarecalledrecombinationsites.

Dependingontheorientationofthe2recombinationsitesonapieceofDNA,threeeventscanoccur:

DNAinsertion（integration）,DNAdeletion（excision）andDNAinversion.

ThesesitescanhaveadirectrepeatofaDNAsequence.ThisresultsinDNAinsertionorDNAdeletion.

Therecombinationsitesareabout20basepairslong.Theyarerecognizedbyanenzymecalledrecombinasethatmediatestherecombination.

1.CSSRcancauseinsertionofaDNAsequence.

2.CSSRcancausedeletionofaDNAsequence.

3.CSSRcancauseinversionofaDNAsequence.

1.Recombinases

1.Thereare2familiesofconservativesite-specificrecombinases:

a.serinerecombinases

b.tyrosinerecombinases

（Serineandtyrosinearebothaminoacids.）

a.SerineRecombinases

Fortheserinerecombinasesthesidechainofaserineinsidetheprotein’sactivesitebreaksaspecificphosphodiesterbondintheDNAattherecombinationsite.

Acovalentprotein-DNAintermediateisproducedbythismechanism.Thisintermediatecontainstheenergyfrombreakingthephosphodiesterbond.ThisallowstheDNAstrandstobejoinedbyareversalofthemechanism.

ForthereverseprocessinwhichtheDNAstrandsarejoined,ahydroxylgroup（OH）fromthecleavedDNAbreaksthecovalentbondholdingtheproteintotheDNA.ThisreleasestherecombinaseandsealstheDNAbreak.

InthiswayeveryDNAbondthatisbrokenduringthereactionisresealedbytherecombinase.

Also,noextraenergyisrequiredforcuttingorjoiningtheDNA.

AsimilarmechanismisusedbyDNAtopoisomerasesandSpo11tobreakandrejoingDNA.

WhatdoDNAtopoisomerasesdo?

removesupercoilingfromDNAduringDNAreplication

WhatdoesSpo11do?

cutsDNAtoinitiatehomologousrecombinationduringmeiosis

Arecombinationsiteisdouble-strandedDNA,soduringrecombination,thereare4strandsofDNAthatmustbecutandrejoinedtocauserecombination.

Theserinerecombinasecutsall4strandsbeforestrandexchangeoccurs.Onerecombinasesubunitcutseachstrand,sotherecombinaseisatetramerof4subunits.

RecombinationoccurswhenanexchangejoinsthedoublestrandedendsofoneDNAmoleculewiththoseoftheotherDNAmolecule.

b.Tyrosinerecombinases

TyrosinerecombinaseshaveasimilarmechanismforcuttingandjoiningtheDNA.

Intyrosinerecombinases,thesidechainofatyrosineintheactivesitebreaksthephosphodiesterbond.

Intyrosinerecombinases,atyrosineintheactivesitebreaksthephosphodiesterbond.

Theserinerecombinasescutall4DNAstrandsbeforeanystrandsarejoined.Incontrast,thetyrosinerecombinasescutandrejoin2strandsbeforecuttingthesecond2strands.

Afterthefirst2strandsarecut,strandexchangeoccurssothataHollidayjunctionisformed.

Thenthesecond2strandsarecut.StrandexchangealsooccurswiththesestrandstoremovetheHollidayjunction.

Similartotheserinerecombinases,foursubunitsofthetyrosinerecombinasearerequiredaseachsubunitcutsasinglestrand.

2.FunctionsofSite-SpecificRecombination

Conservativesite-specificrecombinationisusedforavarietyoffunctionswithincellsandviruses.

a.integrationofvirusDNAintoahostchromosome.

b.generearrangement

Aclassicexampleofconservativesitespecificrecombinationoccursduringlambdaphageinfectionwhenalambdabacteriophageintegratesintothebacterialgenome.

Thereare2waysthatbacteriophagecansurviveafterinfectingabacteria.

1.lyticcycle:

usingthebacterialenzymestoproducenewphageandthenlysisofthebacteriatoreleasethephage.

2.lysogeniccycle:

integrationofthephageDNAintothehostgenome

OnlysomedoublestrandedDNAphagecansurvivebythelysogeniccycle.Allotherphageenterthelyticcycle.Phagethatcanonlyusethelyticcyclearecalledvirulent.

Phagethatcanenterthelysogeniccyclearecalledtemperate.

a.lyticcycle

Thestepsinthelyticcycleare:

1.attachmentofthephagetospecificreceptorsonthebacterium

2.injectionoftheviralnucleicacid

3.ChangingthebacteriasothatitnolongerreplicatesortranscribesitsDNA.

4.Synthesisofphagenucleicacidandproteinsthroughthetranscriptionandtranslationofphagegenes.

5.Assemblyofthephage.Thisiscalledmorphogenesis.Structuralproteinsarerequiredtopackagethenucleicacidintophageproteinsandcatalyticproteinsmediatetheassembly,butarenotpartofthefinalphage.

6.Releaseofthenewphagethroughlysisofthebacterium.Aphageproteincalledlysozymeorendolysinbreaksopenthecellmembrane.

b.lysogeniccycle

Thereare2kindsoflysogeniccycles.

λ（lambda）phageisanexampleofthemostcommontypeinwhichthephageDNAintegratesintothebacterialDNA.

InthelesscommontypeofwhichP1isanexample,thephageDNAreplicatesasaplasmid.

b.lysogeniccycle

Stepsinλphagelysogeniccycle:

1.attachmentofthephagetospecificreceptorsonthebacterium

2.injectionoftheviralnucleicacid

3.Transcriptionandtranslationofanintegrationenzymeandarepressor.

4.TheviralDNAisintegratedintothebacterialchromosomeandiscalledaprophage.

5.ThebacteriumreplicatesandtheprophageisreplicatedwiththebacterialDNA.

Integrationoflambdaphageoccursthroughconservativesite-specificrecombination（CSSR）.

Tointegratethelambdaintegraseprotein（λInt）catalyzesrecombinationbetween2specificsitesnamedattachmentsitesoratt.

TheattPsiteisinthephageDNA,whiletheattBsiteisinthebacterialchromosome.

Lambdaintegrase（λInt）isatyrosinerecombinasethatrecognizesattPandattB.

Anotherproteincalledintegrationhostfactor（IHF）isalsorequiredforintegration.IHFbindstospecificDNAsequencesandcausesabendintheDNAthatallowsλInttobindto2DNAsequences.

Integrationhostfactor（IHF）causesasharpbendintheDNA.

λIntbindsto2DNAsequencesandcutstheDNA.

b.lysogeniccycle

Abacteriumwithaprophagecannotbereinfectedbythesametypeofbacteriophage.IfthephageDNAisinjectedintothebacteria,itisnotreplicated,integratedortranscribed.

Aprophagecaninitiatealyticcycle.Thisiscalledinduction.

Induction

ThelysogeniccycleoccurswhentheactivatorcIIstimulatestranscriptionofcI,whichencodestheλrepressor.Theλrepressorinhibitstranscriptionoflyticgenes.

CertainenvironmentalconditionssuchasUVirradiationcanstimulateinductionoftheprophage.TheUVirradiationactivatesRecA（fromhomologousrecombinationandtheSOSresponse）.RecAcutstheλrepressorresultingintranscriptionofthelyticgenes.

ExcisionoftheprophagerequiresaproteinnamesXis（forexcision）aswellasλIntandIHF.Xisisproducedwheninductionisstimulatedinlambdaphageandotherwiseitisnotproduced.

XiscausesabendintheDNAsimilartoIHF.

Xisbindingstimulatesexcisionandalsoinhibitsintegration.

ThispreventsthephageDNAfrombeingreintegratedintothehostDNA.

Thus,Xisexpressioninabacteriumcommitsthecelltoinductionandthelyticphase.

λIntandIHFarerequiredforintegration.

λInt,IHF,andXisarerequiredforexcision.

B.Transposition

transposableelement:

asequenceofDNAfoundinbothprokaryotesandeukaryotesthatcanmovefromonelocationtoanotherinthegenome.

Bacterialtransposableelementsarecalledtransposons.

Transposition:

movementofatransposableelementtoanewsiteinthegenome

Theenzymethatcatalyzestranspositioniscalledtransposase.

Thereare3kindsoftransposableelementsbasedontheirstructureandmethodoftransposition:

DNAtransposons

viral-likeretrotransposons

Poly-Aretrotransposons

1.DNAtransposons

DNAtransposonsremainasDNA,whilebothviral-likeretrotransposonsandpoly-AretrotransposonsreplicatewithanRNAintermediate.

ADNAtransposonhasinvertedrepeatsattheendsandthecodingsequencefortheenzyme,transposase.

Theinvertedrepeatsvaryfromabout25basepairstohundredsofbasepairs.

ThistypicalDNAtransposonhasinvertedrepeatsandagenefortransposase.Italsohastargetsiteduplications.

Viral-likeretrotransposonshavelongterminalrepeats（LTR）.

Thisoneencodesintegraseandreversetranscriptase（RT）.RTisneededbecauseanRNAintermediateisproducedduringreplication.

3.Poly-Aretrotransposons

Thethirdtypeoftransposableelementisthepoly-Aretrotransposons.

Poly-Aretrotransposonsdonothaverepeatsattheends.Insteadthereisa5’untranslatedregion（UTR）anda3’UTR.Asequenceofpoly-A’sisafterthe3’UTR.

Thereareshorttargetsiteduplicationsoutsideofthe5’and3’UTRs.

Poly-Aretrotransposonshave2genesknownaso