分子英文文献Word格式.docx
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分子英文文献Word格式.docx
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Science.2009November13;
326(5955):
948.
doi:
10.1126/science.1170633.PMCID:
PMC2819049
NIHMSID:
NIHMS169163
CopyrightnoticeandDisclaimer
HowTelomeresSolvetheEnd-ProtectionProblem
TitiadeLange
TitiadeLange,LaboratoryofCellBiologyandGenetics,RockefellerUniversity,NewYork,NY10021,USA.
TitiadeLange:
delange@mail.rockefeller.edu
Thepublisher'
sfinaleditedversionofthisarticleisavailablefreeatScience
SeeotherarticlesinPMCthatcitethepublishedarticle.
Abstract
TheendsofeukaryoticchromosomeshavethepotentialtobemistakenfordamagedorbrokenDNAandmustthereforebeprotectedfromcellularDNAdamageresponsepathways.Otherwise,cellsmightpermanentlyarrestinthecellcycle,andattemptsto“repair”thechromosomeendswouldhavedevastatingconsequencesforgenomeintegrity.Thisend-protectionproblemissolvedbyprotein-DNAcomplexescalledtelomeres.Studiesofmammaliancellshaverecentlyuncoveredthemechanismbywhichtelomeresdisguisethechromosomeends.ComparisontounicellulareukaryotesrevealskeydifferencesintheDNAdamageresponsesystemsthatinadvertentlythreatenchromosomeends.Telomeresappeartobetailoredtothesevariations,explainingtheirvariablestructureandcomposition.
Ofthethreemajorquestionsintelomerebiology,twoweresolvedinthe1980s.First,thenatureoftheDNAsequencesthatconfertelomerefunctionontochromosomeendswasrevealedwhenBlackburnandSzostakshowedthattheshortG-richrepeatsfromtheendsofyeastchromosomesweresufficienttostabilizealinearplasmid(1,2).SincethenithasbecomeclearthatG-richrepeatscaptheendsofmosteukaryoticchromosomes,includingmammalianchromosomesthatendinTTAGGGrepeats.Second,themechanismbywhichtelomericDNAismaintainedwasresolvedwhenBlackburnandGreidershowedthattelomericDNAissynthesizedbytelomerase.Telomeraseisareversetranscriptasethataddstelomericrepeatstothe3′endsofeachchromosome(3).Indoingso,telomerasemakesupfortheshortcomingsofsemiconservativeDNAreplication,whichcannotcompletethesynthesisofchromosomeends.Othersolutionstothisend-replicationproblemexist,notablyinDrosophilaandotherdipterans,butitisnowclearthattelomeraseisthemainmethodbywhicheukaryotesavoidsequencelossattheendsoftheirchromosomes.Ithasbeensuggestedthatearlyeukaryotesusedaprimitiveformoftelomereswithouttelomerasetosolvetheend-replicationproblem(4).Thelateracquisitionoftelomerasenotonlysolvedtheend-replicationproblembutensuredthepresenceofthesamesequenceatallchromosomeends.Oncealltelomeresinthecellhadthesamesequence,telomericDNAbindingfactorscouldevolve,therebyenablingcellstodistinguishnaturalchromosomeendsfromsitesofDNAdamage.
TheEnd-ProtectionProblem
Researchonthethirdmajorissueintelomerebiology,howtelomeressolvetheend-protectionproblem,stagnateduntilthe1990s.Theend-protectionproblemfirstsurfacedearlylastcentury,whenMullerandMcClintockobservedacriticaldistinctionbetweenthebehaviorofbrokenchromosomeendsandtelomeres.Mullerfoundthatchromosomeslackingtheirnaturalendswereunstable;
McClintockdocumentedthepropensityofbrokenends,butnottelomeres,tofuse.However,thefullextentoftheend-protectionproblemremainedobscureuntiltheprinciplesoftheDNAdamageresponsewererevealedinthe1980s.ThefirstinsightcamewhenSzostak,Rothstein,andOrr-WeaverfoundthatlinearDNAintroducedintoeukaryoticcellsisunstablebecausetheDNAendsrecombinewiththegenome(5).ItisnowclearthatintroducedlinearDNAfallsvictimtotwoimportantDNArepairpathwaysthatmendbrokenchromosomes:
homology-directedrepair(HDR)andnonhomologousendjoining(NHEJ).TheobservationthatDNAends(alsoknownasdouble-strandbreaks)areprocessedbytheseDNArepairreactionsraisedthequestionofwhetherthenaturalendsofchromosomesarealsoattackedbyHDRandNHEJ,andifnot,whynot.AsecondquestionarosefromtheworkofHartwellandWeinert,whofoundthatbuddingyeastlackingtheRAD9genefailedtoarrestthecelldivisioncycleinresponsetodouble-strandbreaks(6).Thisexperiment,andearlierobservationsonfissionyeastandmammaliancells(7),revealedthatthecellcyclearrestnormallyassociatedwithDNAdamageisnotduetotheDNAdamageitself.Rather,cellsarrestbecauseoftheactivationofapathwaythatdetectsDNAdamageandblockscellcycleprogressioninresponse.Why,then,arethesepathwaysnotactivatedbythenaturalendsoflinearchromosomes?
ThesefindingsonhoweukaryotesrespondtoDNAdamageshapedthecurrentmoleculardefinitionoftheend-protectionproblem:
HowdotelomerespreventtheactivationoftheDNAdamagesignalingpathways,andwhyaretheyresistanttotherepairpathwaysthatactonDNAends?
Inthecontextofmammaliancells,theend-protectionproblemcanberephrasedinmorepreciseterms,basedoncurrentknowledgeofthemolecularpathwaysthatrecognizeandrepairdouble-strandbreaks(Fig.1).Mammaliancellshavetwoindependentsignalingpathwaysthatareactivatedbydouble-strandbreaks:
(i)theATM(ataxiatelangiectasiamutated)kinasepathway,whichisactivateddirectlybyDNAends,and(ii)theATR(ataxiatelangiectasiaandRad3related)kinasepathway,whichisactivatedbythesingle-strandedDNAformedwhenthe5′endofadouble-strandbreakgetstrimmedback,orresected.Solutionstotheend-protectionproblemmustincludemechanismsthatkeepbothkinasesdormantattelomeres,becausemammaliantelomereshavefeatures(bothaDNAendandaconstitutiveregionofsingle-strandedDNA)thatcouldactivateATMandATR.Asecondsetofreactionsalsoneedstobeblockedattelomeres:
Mammaliancellscanrepairdouble-strandbreaksviaeitherHDRorNHEJ,andthereforeamechanismmustexistthatallowstelomerestoavoidthesereactions.
Fig.1
Theend-protectionproblem.Whenamammalianchromosomebreaks(top),theexposedDNAendscanactivatetwosignalingpathways(theATMandATRkinasepathways)thatarrestthecelldivisioncycleandcaninducecelldeath.ThebrokenchromosomeisusuallyrepairedbyoneoftwodifferentDNArepairpathways(NHEJandHDR),allowingcellstocontinuetheirdivisionswithanintactgenome.ThepresenceoftheseDNAdamageresponsepathwaysposesaproblemfortheendsoflinearchromosomes(telomeres,bottom)becauseactivationofDNAdamagesignalingorDNArepairattelomereswouldbedisastrous.Mammaliantelomeressolvethisend-protectionproblemthroughtheuseofatelomere-specificproteincomplex(shelterin)andanalteredstructure(thet-loop)thattogetherensurethatallfourpathwaysremainblocked.
Theend-protectionproblemofmammalianchromosomesthusinvolvesescapingthepotentialharmfuleffectsoffourdifferentpathways(Fig.1).Failuretodosowillresultincellcyclearrest(underthecommandofATMand/orATR),chromosomeend-to-endfusions(aproductofNHEJ),orsequenceexchanges(mediatedbyHDR)thatinvolvetwotelomeresoratelomereandanotherpartofthegenome.
HowShelterinSolvestheEnd-ProtectionProbleminMammals
Mammaliantelomeressolvetheend-protectionproblemthroughtheagencyofasix-subunitproteincomplexcalledshelterin(8)(Fig.2).ShelterinisendowedwithspecificityfortelomeresthroughtheDNAsequencepreferenceofseveralDNAbindingproteinsinthecomplex.Twoshelterinsubunits,TRF1andTRF2,bindtotheTTAGGGsequencesindouble-strandedDNA,andonesubunit,POT1,bindstothesesequencesinsingle-strandedform.BecausethesethreeproteinsareheldtogetherbyTIN2andTPP1,theselectivityofshelterinfortelomericDNAisexquisite.
Fig.2
Mammaliantelomeres.HumanandmousetelomeresarecomposedoflongstretchesoftherepetitivesequenceTTAGGGandatelomere-specificproteincomplex,shelterin(upperleft).ShelterinderivesitsspecificityfortelomericDNAfromthreeDNAbindingproteins(lowerleft).TRF1andTRF2aretwosimilarproteinsthatbindtothedouble-strandedtelomericrepeatswhilePOT1interactswithTTAGGGrepeatsinsingle-strandedform.TIN2andTPP1connectPOT1toTRF1andTRF2.Rap1isboundtoTRF2.Telomeresarefoundinalariatconformation(upperright),thet-loop,whichresultsfromthestrandinvasionofthe3′single-strandedoverhangintothedouble-strandedtelomericDNA.Shelterinissufficientlyabundanttocovermostofthedouble-strandedtelomericDNA,andthereissufficientPOT1tocoversingle-strandedtelomericDNAeitherinthe3′overhangorintheDloop.Telomeresalsocontainnucleosomesandnumerousshelterin-associatedproteins(notshown).
Thelogicofthemammaliantelomeresystemisthattherepeatssynthesizedbytelomerasefunctionasbindingsitesforshelterin.Asaconsequence,shelterinaccumulatesatallnaturalchromosomeends,whereitpreventstheactivationoftheDNAdamageresponse.Inturn,shelterinisthoughttoberequiredfortherecruitmentoftelomerase(9),ensuringthatthisenzymedoesnotaddtelomericDNAtobrokenendsthatlackshelterinbindingsites.Thesequencespecificityofshelteriniscritical:
Ifitaccumulatedatchromosome-internalsites,itcouldinterferewiththenormalstepsoftheDNAdamageresponseincaseoflocaldamage,anditmightpromoteinappropriate“healing”ofthebrokenendsbytelomerase.TherepressionoftheATMkinasepathwayattelomeresistheassignmentoftheTRF2subunit(Fig.3A).LossofTRF2leadstoactivationoftheATMkinaseatthenaturalendsofmouseorhumanchromosomes(10,11).Thec
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