生物学导论翻译.docx
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生物学导论翻译.docx
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生物学导论翻译
Molecular
mechanisms
ofDNAdouble-
strandbreakrepair
RolandKanaar,JanH.J.Hoeijmakers
andDikC.vanGen
DNAdouble-strandbreaks(DSBs)aremajorthreatstothegenomicintegrityofcells.Ifnottakencareofproperly,theycancausechromosomefragmentation,lossandtranslocation,possiblyresultingincarcinogenesis.UponDSBformation,cell-cycle
checkpointsaretriggeredandmultipleDSBrepairpathwayscanbeactivated.RecentresearchontheNijmegenbreakagesyndrome,whichpredisposespatientstocancer,suggestsadirectlinkbetweenactivationofcell-cyclecheckpointsandDSBrepair.Furthermore,thebiochemicalactivitiesofproteinsinvolvedinthetwomajorDSB
repairpathways,homologousrecombinationandDNAend-joining,arenowbeginningtoemerge.ThisreviewdiscussesthesenewfindingsandtheirimplicationsforthemechanismsofDSBrepair.TheintegrityofDNAinsidecellsisconstantlybeingchallengedbyendogenousandexogenousDNA-damagingagents.BecausealargevarietyoflesionscanoccurinDNA,itisnotsurprisingthatmultiplepathwayshaveevolvedthateachrepairasubsetoftheselesions
1
.ThesignificanceofDNArepairisillustratedbythephenotypesofxerodermapigmentosum,Cockayne’ssyndrome,trichothiodystrophyandhereditarynonpolyposiscolorectalcancerpa2,3.ThesedisordersarecausedbymutationsinDNArepairgenesthatpredisposethepatientstocancer,neurologicalabnormalitiesorboth.Inaddi-tiontoefficientDNArepair,correctactivationofcell-cyclecheckpointsuponinductionofDNAdamageisofcrucialimportanceforthemaintenanceofgenomicintegrity.CheckpointsallowactivelydividingcellstopauseandrepairDNAdamagebeforesegregationofthereplicatedgenomeintodaughtercells.TheirimportanceisunderscoredbyinheriteddisordersassociatedwithdefectsinactivatingcellcyclecheckpointssuchasataxiatelangiectasiaandNijmegenbreakagesyndrome4,5.ThesedisorderscausehypersensitivitytoDNA-damagingagentsandspontaneouschromosomalinstability.
Inthisreview,wefocusonmechanismsofDNAdouble-strandbreak(DSB)repair.DSBsaregeneratedbyendogenouslyproducedradicalsandexogenousagentssuchasionizingradiation(IR),whichisoftenusedinanti-cancertherapy.RepairofDSBs
isofcardinalimportancetopreventchromosomalfragmentation,translocationsanddeletions.Inthesoma,thegenomicinstabilityresultingfrompersistentorincorrectlyrepairedDSBscanleadtocarcinogenesisthroughactivationofoncogenes,inactivationoftumour-suppressorgenesorlossofheterozygosity,whileinthegermlinetheycanleadtoinborndefects.
ThedeleteriouseffectsofDSBshavetriggeredtheevolutionofmultiplepathwaysfortheirrepair6,7.Homologousrecombinationrequiresextensivere-
gionsofDNAhomologyandrepairsDSBsaccuratelyusinginformationontheundamagedsisterchromatidorhomologouschromosome.DNAend-joining,ontheotherhand,usesno,orextremelylimited,sequencehomologytorejoinjuxtaposedendsinamannerthatneednotbeerrorfree(Figs1and2;see
Table1forasummaryofthepropertiesoftheproteinsinvolvedandthecorrespondingmutantphenotypes).GeneticanalysesoftheeffectsofIRonSaccharomycescerevisiaeandrodentcellshavebeencrucialinidentifyinggenesinvolvedinDSByeastmutantsweredefectiveinhomologousrecombination,whereasthemutantrodentcelllinesweredefectiveinDNAend-joining.However,recentstudieshaveshownthatbothpathwayscontribute
toDSBrepairinyeastandmammalsbutthattheirrelativecontributioncanvary(seebelow).Thegeneticanalysesarenowbeingcomplementedby
thebiochemicalcharacterizationoftheDSBrepairpathways.DSBrepairbyhomologousrecombinationGeneticanalysesinS.cerevisiaehaveledtotheidentificationofgenesrequiredforDSBrepairbyhomologousrecombination9,12.Screensforradiation-sensitiveormeioticrecombination-deficientyeastcellshaverevealedtheinvolvementoftheRAD52epistasisgroupgenes(RAD50,RAD51,RAD52,RAD54,RAD55,RAD57,RAD59,MRE11andXRS2).TheimportanceoftheRAD52recombi-
nationalDNArepairpathwayisunderscoredbyitsevolutionaryconservation.MouseandhumangeneswithsequencesimilaritytoRAD50,RAD51,RAD52,
RAD54andMRE11havebeenisolated7,10.Theavail-abilityoftheRAD52groupgeneshasprovidedtheessentialtoolsforbiochemicalanalysesofthe
molecularmechanismofhomologousrecombination.TheaminoacidsequenceofS.cerevisiaeRad51pguidedtheinitialbiochemicalexperimentsbecause
ofitssimilaritytotheEscherichiacolirecombinationproteinRecA6.Theseexperimentsshowedthatkeyreactionsinhomologousrecombination–thesearch
forhomologousDNAandDNAstrandexchangeareperformedbyremarkablysimilarmechanismsinbacteria,yeastandhumancells.RecA,Rad51pandhumanRAD51polymerizeonDNAtoformanucleoproteinfilamentthatsearchesforhomologousDNA13.ThissearchendswiththeformationofjointmoleculesbetweenthehomologousDNAs,followedbyexchangeofDNAstrands(seeFig.1).TheS.cerevisiaeproteinsRad55pandRad57pdisplaysequencesimilaritytoRad51p.However,itisunlikelythatthesearefunctionalhomologuesofRad51psincethereisnoevidenceforRad55por
Rad57pnucleoproteinfilamentformation.Instead,thesetwoproteinsformaheterodimerthatcanstimulateRad51p-mediatedstrandexchangeatsub-
stoichiometricamountsrelativetoRad51p14.ThereareseveralotherproteinswithsimilaritytoRad51pinmammalsaswell.Sincedisruptionofmouse
RAD51resultsinembryoniclethality6,theothermammalianproteinswithsequencesimilaritytoRAD51(RAD51B,RAD51C,RAD51D,XRCC2andlossofmouseRAD51functionXRCC3)areapparentlyunabletosubstituteforthe
15–21.Atpresent,itisunclearwhethertheseproteinscanformnucleopro-
einfilaments,likeRAD51,orwhethertheyfunctionanalogouslytoRad55/57p.ThereareseveralreasonswhymultipleRAD51-likeproteinsthatcanformnucleoproteinfilamentsmightberequiredinmammals.•Theycouldassembleseparatenucleoproteinfila-mentsthathavespecificcharacteristicstofacilitaterepairinpartsofthegenomethatdifferinchromatinstructureorcontainspecialsequencessuchastelomeresorcentromeres.Inaddition,nucleoproteinfilamentswithslightlydifferentpropertiesmightberequiredtorepairdifferenttypesofDNAdamage,suchasDSBsorinterstrandDNAcrosslinks.
•Theycouldformmixedfilamentsinwhichthedifferentproteinshavespecificfunctions.Forexample,aRAD51-likeprotomercouldbeincor-
poratedintheRAD51nucleoproteinfilamentatsubstoichiometriclevelstoserveasadockingsiteforotherproteinsthatneedtointeractwiththefilament,butnotwitheveryRAD51protomer.
•Theycouldberequiredwhenthehomologouschromosomeinsteadofthesisterchromatidisusedasatemplate.
•Theproteinscouldhavetissue-specificfunctions.ThestepsthatleadtoS.cerevisiaeRad51p-mediatedjoint-moleculeformationcanbestimulatedby
overcomeinhibitoryeffectsofcompetingreactionsinthecell.InadditiontoRad55/57p,Rad52pplaysacrucialroleinstimulatingRad51p.Biochemical
analysesofRad52phaveshownthatitpromotesannealingofcomplementarysingle-strandedDNAs22andfunctionsasamediatorbetweenRad51pandthe
single-strandDNA-bindingproteinRP-Atostimu-lateRad51p-mediatedDNAstrandexchange14,23,24Similarly,humanRAD52proteinassistshumanRAD51informingjointmolecules25.However,thephenotypeofRAD52mutationsinS.cerevisiae,fissionyeastSchizosaccharomycespombeandmousecellsdiffersdramatically.WhiletheefficiencyofrecombinationisreducedbymorethanthreeordersofmagnitudeintheS.cerevisiaerad52mutant,itisonlytwo-foldlowerinthecorrespondingS.pombemutantandinmouseRAD52-knockoutembryonicstemcells7,26.TheproteinencodedbytheS.cerevisiaeRAD59geneshowssequencesimilaritytoRad52p
27.AdditionalhomologuesofRAD52andRAD59havenotyetbeenidentifiedinotherspecies,buttheexistenceofRAD59homologuesinotherspeciescouldhelpexplainthedifferenceinphenotypesconferredbyRAD52ifsomeofthefunctionsofRAD52canalsobeassumedbyRAD59inS.Pombeandmammals.
Anotherkeycomponentofthehomologous-recombinationmachinery,theDNA-dependentATPaseRad54p28,29,belongstotheSNF2–SWI2proteinfamily6
.MembersofthisfamilyareinvolvedinmanyastsofDNAmetabolism,suchastranscription,recombinationandDNArepair.GeneticexperimentshaveshownthatRAD54-knockoutyeast,chickenandmousecellsareIR-sensitiveandhaveareducedlevelofhomologousrecombination12,30,31.Rad54pcaninteractwithRad51p32–34,andbiochemicalexperi-mentshaveshownthatRad54pstimulatesRad51pmediatedjoint-moleculeformation28.BecauseRad51pactivitycanbestimulatedbyanumberofproteins(Rad52p,Rad54pandRad55/57p),itisofinteresttodeterminetheexactstepinthestrand-exchangereactionatwhichtheseproteinsact.TheotherproteinsintheRAD52epistasisgroup,Rad50p,Mre11pandXrs2p,formacomplexthatisrequiredfortheformationofmeiosis-specifiDSBs,whicharesubsequentlyrepairedthroughrecombinationwiththehomologouschromosome9.Inmitoticcells,thecomplexisprobablyinvolvedinonlyasubsetofDNArepairreactionsthat
requirehomologousrecombination35.Interestingly,recentexperimentshaveshownthatRad50p,Mre11pandXrs2parealsorequiredforDNAend-joining(seebelow).DSBrepairbyDNAend-joiningTheDNAend-joiningpathwayofDSBrepairwasfirstcharacterizedinrodentcells36.UsingacollectionofChinesehamsterovarycelllinesselectedonhebasiso
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