基因的分子生物学RecombinationandRepair

2007-04-27115RECOMBINATIONANDREPAIR2007-04-272Homologousrecombination(generalizedrecombination)involvesareciprocalexchangeofsequencesofDNA,e.g.betweentwochromosomesthatcarrythesamegeneticloci.Site-specificrecombination(Specializedrecombination)occursbetween2specificsequences,asinphageintegration/excisionorresolutionofcointegratestructuresduringtransposition.Transpositionreferstothemovementofatransposontoanewsiteinthegenome.Copychoice

isatypeofrecombinationusedbyRNAviruses,inwhichtheRNApolymeraseswitchesfromonetemplatetoanotherduringsynthesis.Figure15.1

GeneralizedrecombinationcanoccuratanypointalongthelengthsoftwohomologousDNAs.Figure15.2FigureIntroduction2007-04-273Abivalent

isthestructurecontainingallfourchromatids(tworepresentingeachhomologue)atthestartofmeiosis.Synapsis(联会)

describestheassociationofthe2pairsofsisterchromatids(representinghomologouschromosomes)thatoccursatthestartofmeiosis;theresultingstructureiscalledabivalent.Chromosomepairing

isthecouplingofthehomologouschromosomesatthestartofmeiosis.Thesynaptonemalcomplex

describesthemorphologicalstructureofsynapsedchromosomes.Breakageandreunion

describesthemodeofgeneticrecombination,inwhichtwoDNAduplexmoleculesarebrokenatcorrespondingpointsandthenrejoinedcrosswise(involvingformationofalengthofheteroduplexDNAaroundthesiteofjoining).Achiasma(交叉)

(pl.chiasmata)isasiteatwhichtwohomologouschromosomesappeartohaveexchangedmaterialduringmeiosis.Figure15.4

Recombinationoccursduringthefirstmeioticprophase.Thestagesofprophasearedefinedbytheappearanceofthechromosomes,eachofwhichconsistsoftworeplicas(sisterchromatids),althoughtheduplicatedstatebecomesvisibleonlyattheend.Themolecularinteractionsofanyindividualcrossing-overeventinvolvetwoofthefourduplexDNAs.4.15.2Homologousrecombinationoccursbetweensynapsedchromosomes2007-04-274HeteroduplexDNA(HybridDNA)isgeneratedbybasepairingbetweencomplementarysinglestrandsderivedfromthedifferentparentalduplexmolecules;itoccursduringgeneticrecombination.AHolliday

structureisanintermediatestructureinhomologousrecombination,wherethetwoduplexesofDNAareconnectedbythegeneticmaterialexchangedbetweentwoofthefourstrands,onefromeachduplex.AjointmoleculeissaidtoberesolvedwhennicksinthestructurerestoretwoseparateDNAduplexes.Splicerecombinant

DNAresultsfromaHollidayjunctionbeingresolvedbycuttingthenon-exchangedstrands.BothstrandsofDNAbeforetheexchangepointcomefromonechromosome;theDNAaftertheexchangepointcomefromthehomologouschromosome.Patchrecombinant

DNAresultsfromaHollidayjunctionbeingresolvedbycuttingtheexchangedstrands.Theduplexislargelyunchanged,exceptforaDNAsequenceononestrandthatcamefromthehomologouschromosome.4.15.3BreakageandreunioninvolvesheteroduplexDNA2007-04-275Figure15.5

Recombinationbetween2pairedduplexDNAscouldinvolvereciprocalsingle-strandexchange,branchmigration,andnicking.Figure15.6

Branchmigrationcanoccurineitherdirectionwhenanunpairedsinglestranddisplacesapairedstrand.Figure15.7

ResolutionofaHollidayjunctioncangenerateparentalorrecombinantduplexes,dependingonwhichstrandsarenicked.BothtypesofproducthavearegionofheteroduplexDNA.2007-04-276Adouble-strandbreak(DSB)occurswhenbothstrandsofaDNAduplexarecleavedatthesamesite.Geneticrecombinationisinitiatedbydouble-strandbreaks.Thecellalsohasrepairsystemsthatactondouble-strandbreakscreatedatothertimes.Figure15.8

Recombinationisinitiatedbyadouble-strandbreak,followedbyformationofsingle-stranded3’ends,oneofwhichmigratestoahomologousduplex.4.15.4Double-strandbreaksinitiaterecombination2007-04-277Thesynaptonemalcomplex

describesthemorphologicalstructureofsynapsedchromosomes.Anaxialelement

isaproteinaceousstructurearoundwhichthechromosomescondenseatthestartofsynapsis.Alateralelement

isastructureinthesynaptonemalcomplex.Itisanaxialelementthatisalignedwiththeaxialelementsofotherchromosomes.Thecentralelement

isastructurethatliesinthemiddleofthesynaptonemalcomplex,alongwhichthelateralelementsofhomologouschromosomesalign.Recombinationnodules(Node)aredenseobjectspresentonthesynaptonemalcomplex;theymayrepresentproteincomplexesinvolvedincrossing-over.Figure15.10

Eachpairofsisterchromatidshasanaxismadeofcohesins.Loopsofchromatinprojectfromtheaxis.Thesynaptonemalcomplexisformedbylinkingtogethertheaxesviazipproteins.4.15.5Recombiningchromosomesareconnectedbythesynaptonemalcomplex2007-04-2784.15.6Thesynaptonemalcomplexformsafterdouble-strandbreaksFigure15.11

Spo11iscovalentlyjoinedtothe5’endsofdouble-strandbreaks.Figure15.12

Double-strandbreaksappearwhenaxialelementsform,anddisappearduringtheextensionofsynaptonemalcomplexes.JointmoleculesappearandpersistuntilDNArecombinantsaredetectedattheendofpachytene(粗线期).2007-04-279·TheRecBCDcomplexhasnucleaseandhelicasesactivities.·ItbindstoDNAdownstreamofachisequence,unwindstheduplex,anddegradesonestrandfrom3’–5’asitmovestothechisite.·ThechisitetriggerslossoftheRecDsubunitandnucleaseactivity.Figure15.13

RecBCDnucleaseapproachesachisequencefromoneside,degradingDNAasitproceeds;atthechisite,itmakesanendonucleolyticcut,losesRecD,andretainsonlythehelicaseactivity.4.15.8ThebacterialRecBCDsystemisstimulatedbychisequences2007-04-2710Figure15.14

RecApromotestheassimilationofinvadingsinglestrandsintoduplexDNAsolongasoneofthereactingstrandshasafreeend.Figure15.15

RecA-mediatedstrandexchangebetweenpartiallyduplexandentirelyduplexDNAgeneratesajointmoleculewiththesamestructureasarecombinationintermediate.Single-strandassimilation(Single-stranduptake)describestheabilityofRecAproteintocauseasinglestrandofDNAtodisplaceitshomologousstrandinaduplex;thatis,thesinglestrandisassimilatedintotheduplex.4.15.9Strand-transferproteinscatalyzesingle-strandassimilation(同化)2007-04-2711·TheRuvcomplexactsonrecombinantjunctions.·RuvArecognizesthestructureofthejunctionandRuvBisahelicasethatcatalyzesbranchmigration.·RuvCcleavesjunctionstogeneraterecombinationintermediates.Figure15.17

BacterialenzymescancatalyzeallstagesofrecombinationintherepairpathwayfollowingtheproductionofsuitablesubstrateDNAmolecules.Figure15.16

RuvABisanasymmetriccomplexthatpromotesbranchmigrationofaHollidayjunction.4.15.10TheRuvsystemresolvesHollidayjunctions2007-04-2712SupercoilingdescribesthecoilingofaclosedduplexDNAinspacesothatitcrossesoveritsownaxis.Figure15.20

SeparationofthestrandsofaDNAdoublehelixcouldbeachievedinseveralways.4.15.12SupercoilingaffectsthestructureofDNA2007-04-2713AtypeItopoisomerase

isanenzymethatchangesthetopologyofDNAbynickingandresealingonestrandofDNA.AtypeIItopoisomerase

isanenzymethatchangesthetopologyofDNAbynickingandresealingbothstrandsofDNA.Figure15.21

ThetopologicalstructureofDNAischangedduringreplicationandtranscription.Strandseparationforreplication(ortranscription)requiresabaseturnofDNAtobeunwound.TranscriptioncreatespositivesupercoilsaheadoftheRNApolymerase.Replicationofacirculartemplateproducestwocatenateddaughtertemplates.4.15.13TopoisomerasesrelaxorintroducesupercoilsinDNATypeIenzymesactbybreakingasinglestrandofDNA;typeIIenzymesactbymakingdouble-strandbreaks.2007-04-2714Single-strandpassage

isareactioncatalyzedbytypeItopoisomeraseinwhichonesectionofsingle-strandedDNAispassedthroughanotherstrand.Figure15.22

BacterialtypeItopoisomerasesrecognizepartiallyunwoundsegmentsofDNAandpassonestrandthroughabreakmadeintheother.Figure15.23

TypeIItopoisomerasescanpassaduplexDNAthroughadouble-strandbreakinanotherduplex.4.15.14Topoisomerasesbreakandresealstrands2007-04-2715Thesigninversion

modeldescribesthemechanismofDNAgyrase(促旋酶).DNAgyrasebindsapositivesupercoil(inducingacompensatorynegativesupercoilelsewhereontheclosedcircularDNA),breaksbothstrandsinoneduplex,passestheotherduplexthrough,andresealsthestrands.Enzymeturnover

istheprocessthroughwhichtheenzymereturnstoitsoriginalshape,enablingtheenzymetocatalyzeanotherreaction.·E.coligyraseisatypeIItopoisomerasethatusedhydrolysisofATPtoprovideenergytointroducenegativesupercoilsintoDNA.Figure15.24

DNAgyrasemayintroducenegativesupercoilsinduplexDNAbyinvertingapositivesupercoil.4.15.15Gyrasefunctionsbycoilinversion2007-04-2716Site-specificrecombination(Specializedrecombination)occursbetweentwospecificsequences,asinphageintegration/excisionorresolutionofcointegratestructuresduringtransposition.Prophage

isaphagegenomecovalentlyintegratedasalinearpartofthebacterialchromosome.Integration

ofviraloranotherDNAsequencedescribesitsinsertionintoahostgenomeasaregioncovalentlylinkedoneithersidetothehostsequences.Theexcision

stepinanexcision-repairsystemconsistsofremovingasingle-strandedstretchofDNAbytheactionofa5’-3’exonuclease.Asecondaryattachment

site

isalocusonthebacterialchromosomeintowhichphagelambdaintegrateinefficientlybecausethesiteresemblestheattsite.Figure15.25

CircularphageDNAisconvertedtoanintegratedprophagebyareciprocalrecombinationbetweenattPandattB;theprophageisexcisedbyreciprocalrecombinationbetweenattLandattR.Gateway®4.15.16Specializedrecombinationinvolvesspecificsites2007-04-2717Figure15.27

Staggered(交错)cleavagesinthecommoncoresequenceofattPandattBallowcrosswisereuniontogeneratereciprocalrecombinantjunctions.4.15.17Site-specificrecombinationinvolvesbreakageandreunion2007-04-27184.15.18Site-specificrecombinationresemblestopoisomeraseactivityFigure15.28

Integrasescatalyzerecombinationbyamechanismsimilartotopoisomerases.StaggeredcutsaremadeinDNAandthe3’-phosphateendiscovalentlylinkedtoatyrosineintheenzyme.ThenthefreehydroxylgroupofeachstrandattackstheP-Tyrlinkoftheotherstrand.ThefirstexchangeshowninthefiguregeneratesaHollidaystructure.Thestructureisresolvedbyrepeatingtheprocesswiththeotherpairofstrands.Figure15.29

AsynapsedloxArecombinationcomplexhasatetramerofCrerecombinases,withoneenzymemonomerboundtoeachhalfsite.Twoofthefouractivesitesareinuse,actingoncomplementarystrandsofthetwoDNAsites.2007-04-2719Anintasome

isaprotein-DNAcomplexbetweenthephagelambdaintegrase(Int)andthephagelambdaattachmentsite(attP).Figure15.30

IntandIHFbindtodifferentsitesinattP.TheIntrecognitionsequencesinthecoreregionincludethesitesofcutting.Figure15.31

MultiplecopiesofIntproteinmayorganizeattPintoanintasome,whichinitiatessite-specificrecombinationbyrecognizingattBonfreeDNA.4.15.19Lambdarecombinationoccursinanintasome2007-04-2720Astructuraldistortion

isachangeintheshapeofamolecule.Apyrimidinedimer

isformedwhenultravioletirradiationgeneratesacovalentlinkdirectlybetweentwoadjacentpyrimidinebasesinDNA.ItblocksDNAreplication.Photoreactivation

usesawhite-light-dependentenzymetosplitcyclobutanepyrimidinedimersformedbyultravioletlight.Mismatchrepair

correctsrecentlyinsertedbasesthatdonotpairproperly.Theprocesspreferentiallycorrectsthesequenceofthedaughterstrandbydistinguishingthedaughterstrandandparentalstrand,sometimesonthebasisoftheirstatesofmethylation.Excisionrepair

describesatypeofrepairsysteminwhichonestrandofDNAisdirectlyexcisedandthenreplacedbyresynthesisusingthecomplementarystrandastemplate.Recombination-repair

isamodeoffillingagapinonestrandofduplexDNAbyretrievingahomologoussinglestrandfromanotherduplex.Error-prone

synthesisoccurswhenDNAincorporatesnoncomplementarybasesintothedaughterstrand.4.15.20RepairsystemscorrectdamagetoDNA2007-04-2721Figure15.32

DeaminationofcytosinecreatesaU-Gbasepair.Uracilispreferentiallyremovedfromthemismatchedpair.Figure15.33

Areplicationerrorcreatesamismatchedpairthatmaybecorrectedbyreplacingonebase;ifuncorrected,amutationisfixedinonedaughterduplex.Figure15.34Ultravioletirradiationcausesdimerformationbetweenadjacentthymines.Thedimerblocksreplicationandtranscription.Figure15.35

Methylationofabasedistortsthedoublehelixandcausesmispairingatreplication.2007-04-2722Figure15.36

DepurinationremovesabasefromDNA,blockingreplicationandtranscription.Figure15.37

RepairgenescanbeclassifiedintopathwaysthatusedifferentmechanismstoreverseorbypassdamagetoDNA.Figure15.38

Excision-repairdirectlyreplacesdamagedDNAandthenresynthesizesareplacementstretchforthedamagedstrand.2007-04-2723Incision(切开)

isastepinamismatchexcisionrepairsystem.AnendonucleaserecognizesthedamagedareaintheDNA,andisolatesitbycuttingtheDNAstrandonbothsidesofthedamage.Theexcision(切除)

stepinanexcision-repairsystemconsistsofremovingasingle-strandedstretchofDNAbytheactionofa5’-3’exonuclease.·TheUvrsystemmakesincisions~12basesapartonbothsidesofdamagedDNA,removestheDNAbetweenthem,andresynthesizesnewDNA.Figure15.39

Excision-repairremovesandreplacesastretchofDNAthatincludesthedamagedbase(s).4.15.21ExcisionrepairsystemsinE.coli2007-04-2724·Uracilandalkylated

basesarerecognizedbyglycosylasesandremoveddirectlyfromDNA.·Pyrimidinedimersarereversedbybreakingthecovalentbondsbetweenthem.·Methylasesaddamethylgrouptocytosine.·Allthesetypesofenzymeactbyflippingthebaseoutofthedoublehelix,where,dependingonthereaction,itiseitherremovedorismodifiedandreturnedtothehelix.Figure15.41

AglycosylaseremovesabasefromDNAbycleavingthebondtothedeoxyribose.Figure15.42

Aglycosylasehydrolyzesthebondbetweenbaseanddeoxyribose(usingH20),butalyasetakesthereactionfurtherbyopeningthesugarring(usingNH2).Figure15.43

Amethylase"flips"thetargetcytosineoutofthedoublehelixinordertomodifyit.4.15.22Baseflippingisusedbymethylasesandglycosylases2007-04-2725Figure15.45

GATCsequencesaretargetsfortheDammethylaseafterreplication.Duringtheperiodbeforethismethylationoccurs,thenonmethylatedstrandisthetargetforrepairofmismatchedbases.4.15.24Controllingthedirectionofmismatchrepair2007-04-2726Figure15.46

MutSrecognizesamismatchandtranslocatestoaGATCsite.MutHcleavestheunmethylatedstrandattheGATC.EndonucleasesdegradethestrandfromtheGATCtothemismatchsite.Figure15.47

TheMutS/MutLsysteminitiatesrepairofmismatchesproducedbyreplicationslippage.2007-04-2727Single-strandexchange

isareactioninwhichoneofthestrandsofaduplexofDNAleavesitsformerpartnerandinsteadpairswiththecomplementarystrandinanothermolecule,displacingitshomologueinthesecondduplex.Figure15.48

AnE.coliretrievalsystemusesanormalstrandofDNAtoreplacethegapleftinanewlysynthesizedstrandoppositeasiteofunrepaireddamage.4.15.25Recombination-repairsystemsinE.coli2007-04-27284.15.26RecombinationisanimportantmechanismtorecoverfromreplicationerrorsFigure15.49

AreplicationforkstallswhenitreachesadamagedsiteinDNA.Branchmigrationoccurstomovetheforkbackward,andthetwodaughterstrandspairtoformaduplex.Afterthedamagehasbeenrepaired,ahelicasemaycauseforwardbranchmigrationtor