大学 生物化学 F4

BIOCHEMISTRYF4DNAREPLICATIONINEUKARYOTES应用化学1201张陇涛1215020129F4DNAREPLICATIONIN

EUKARYOTESIneukaryotes,thecellcycleconsistsofG1,S,G2andMphases.

Most

differencesinthecycletimesofdifferentcellsareduetodifferencesin

thelengthoftheG1phase.QuiescentcellsaresaidtobeintheG0phase.Cellcycle真核生物中，细胞周期包括Gi、S、和M期。不同细胞的不同周期时间主要是由Gi期的长短决定。静止细胞（quiescentcells)认为是处于GQ期。细胞周期DNAreplicationoccursonlyintheSphase.Itoccursatmany

chromosomalorigins,isbi-directionalandsemi-conservative.Setsof

20–80repliconsactasreplicationunitsthatareactivatedinsequence.MultiplerepliconsDNA复制只发生于S期（Sphase)。它发生于染色体上许多起始点，是双向而且是¥保留复制。20〜80个成套的复制（repli-con)依次被激活作为复制单位。多复制子KeyNotesDNApolymerasesαandδreplicatechromosomalDNA,DNA

polymerasesβandεrepairDNA,andDNApolymeraseγreplicates

mitochondrialDNA.FiveDNApolymerasesDNA聚合酶α和δ复制染色体DNA，DNA聚合酶β和ε修复DNA,DNA聚合酶γ复制线粒体DNA。5种DNA聚合酶DNApolymeraseαandδsynthesizethelaggingstrand,viaOkazaki

fragments.TheRNAprimersaresynthesizedbyDNApolymeraseαwhichcarriesaprimasesubunit.DNApolymeraseδsynthesizesthe

leadingstrand.Leadingandlagging

strandsDNA聚合酶α将δ片段合成滞后链，而聚合酶δ合成前导链。RNA引物由DNA聚合酶α来合成，该酶带有引物酶的一个亚基。前导链和滞后链Telomerase,aDNApolymerasethatcontainsanintegralRNAthatacts

asitsownprimer,isusedtoreplicateDNAattheendsofchromosomes

(telomeres).Telomerereplication端粒酶（telomerase)，一种自身含有可作为引物的内在RNA的DNA聚合酶，用于在染色体末端合成DNA(端粒，telomeres)。端粒复制NucleosomesdonotdissociatefromtheDNAduringDNAreplication;

rathertheymustopenuptoallowthereplicationapparatustopass.Both

daughterDNAmoleculeshaveoldhistonesboundtothembutnew

histonesmustalsobesynthesizedtoallowalltheDNAtobepackaged

correctlyinnucleosomes.Replicationof

chromatinDNA复制时核小体不从DNA上解离，但它们必须开放以允许复制体(replicationapparatus)通过。两个子代DNA分子都与先前的组蛋白组合，但还必须合成新的组蛋白使所有DNA在核小体中能正确包装。染色质的复制Relatedtopics

DNAstructure(F1)

DNAreplicationinbacteria(F3)相关主题

DNA结构（F1)

原核生物中DNA的复制(F3)Cellcycle

细胞周期Thelifeofaeukaryoticcellcanbedefinedasacellcycle(Fig.1).MitosisandcelldivisionoccurintheMphasewhichlastsforonlyabout1h.ThisisfollowedbytheG1phase(Gforgap),thentheSphase(Sforsynthesis),duringwhichtimethechromosomalDNAisreplicated,andfinallytheG2phaseinwhichthecells

prepareformitosis.Eukaryoticcellsinculturetypicallyhavecellcycletimesof

16–24hbutthecellcycletimecanbemuchlonger(>100days)forsomecellsin

amulticellularorganism.Mostofthevariationincellcycletimesoccursby

differencesinthelengthoftheG1phase.Somecellsinvivo,suchasneurons,

stopdividingcompletelyandaresaidtobequiescent,lockedinaG0phase.真核细胞的一生可以定义为一个细胞周期（cellcycle)(图F4.1)。有丝分裂和细胞分裂发生在约持续1小时的M期，紧接着是（^期（G代表gap),然后是S期（S代表synthesis)，在此期间染色体DNA被复制，最后是细胞准备有丝分裂的G2期。培养的真核细胞的周期时间通常为16~24小时，但在多细胞机体中一些细胞的周期时间可以很长（>100天）。细胞周期长度的变化大部分发生在(¾期。一些体内细胞，如神经元，完全停止分裂，被认为是静止的，处于仏期（Gcphase)。Multiplereplicons多复制例子Ineukaryotes,replicationofchromosomalDNAoccursonlyintheSphaseof

thecellcycle.AsforbacterialDNA(seeTopicF3),eukaryoticDNAisreplicatedsemi-conservatively.ReplicationofeachlinearDNAmoleculeinachromosomestartsatmanyorigins,oneevery30–300kbofDNAdependingonthespecies

andtissue,andproceedsbi-directionallyfromeachorigin.Theuseofmultiple

originsisessentialinordertoensurethatthelargeamountofchromosomal

DNAinaeukaryoticcellisreplicatedwithinthenecessarytimeperiod.Ateach

origin,areplicationbubbleformsconsistingoftworeplicationforksmovingin

oppositedirections.TheDNAreplicatedunderthecontrolofasingleoriginis

calledareplicon.DNAsynthesisproceedsuntilreplicationbubblesmergetogether(Fig.2).在真核生物中，染色体DNA的复制只发生在细胞周期的S期。与细菌DNA相似（参看F3)，真核生物的DNA也是半保留复制。染色体线性DNA分子的复,制起始于多个起始点，随种系与组织不同，大约每3〜300kbDNA就有一个起始点，而且从每个起始点处双向进行。多起始点的应用对于保证染色体DNA在必需时间内完成复制是至关重要的。在每个起始点，复制泡由两个反向运动的复制叉组成。由一个起始点控制的箄制DNA称为一个复制子。DNA合成不断进行直至复制泡融合在一起(图F4.2)。Fig.1.

Theeukaryoticcellcycle.TheSphaseistypically6–8hlong,G2isaphaseinwhich

thecellpreparesformitosisandlastsfor2–6h,mitosisitself(M)isshortandtakesonlyabout

1h.ThelengthofG1isveryvariableanddependsonthecelltype.CellscanenterG0,

aquiescentphase,insteadofcontinuingwiththecellcycle.图F4.1真核细胞周期。S期通常6~8小时；（¾期是细胞准备有丝分裂的时期，延续2~6小时；有丝分裂（M期）时间短，仅1小时左右；（¾的长短变化很大，依赖于细胞类型。细胞亦可进人Go期即静止期.则不再进人细胞周期Fig.2.

ReplicationofeukaryoticchromosomalDNA.Replicationbeginsatmanyoriginsand

proceedsbi-directionallyateachlocation.Eventuallythereplicationeyesmergetogetherto

producetwodaughterDNAmolecules,eachofwhichconsistsofoneparentalDNAstrand

(thinline)andonenewlysynthesizedDNAstrand(thickline).图F4.2真核生物染色体DNA的复制。复制开始于多个起始点并在'每个位点双向进行。最后这些复制眼融合产生两个子代DNA分子，每个分子都含有一条来自母链的DNA链（细线）和一条新合成的DNA链（粗线）

Alloftheregionsofachromosomearenotreplicatedsimultaneously.Rather,manyreplicationeyeswillbefoundinonepartofthechromosomeandnoneinanothersection.Thusreplicationoriginsareactivatedinclusters,calledreplica-tionunits,consistingof20–80origins.DuringSphase,thedifferentreplication

unitsareactivatedinasetorderuntileventuallythewholechromosomehas

beenreplicated.Transcriptionally-activeDNAappearstobereplicatedearlyinSphase,whilstchromatinthatiscondensedandnottranscriptionallyactiveis

replicatedlater.Multiplereplicons多复制例子

染色体的各部分不是同时进行复制的，而是在染色体的某一部分可以一存在很^复-制服，—而在另=■部分却二■个也没有。因此复制起始点是成簇溻活（activatedinclusters)，称为复制单位（replicationunits)，每个复制单位含有20〜80个起始点。在S期不同的复制单位被依次激活，直到整条染色体完成复制。转录活跃的基因在S期较早被复制，而浓缩的转录不活跃的染色质复制得较迟。Five

DNApolymerases5种DNa聚合酶EukaryoticcellscontainfivedifferentDNApolymerases;α,β,γ,δandε.The

DNApolymerasesinvolvedinreplicationofchromosomalDNAareαandδ.DNApolymerasesβandεareinvolvedinDNArepair.Allofthesepolymerases

exceptDNApolymeraseγarelocatedinthenucleus;DNApolymeraseγis

foundinmitochondriaandreplicatesmitochondrialDNA.真核生物细胞含有5种不同的DNA聚合酶：α,β,γ,δ和ε。在染色体'DNA复制中所用的DNA聚合酶为α和δ。DNA聚合酶β和ε用于DNA修复。除了γ外所有的DNA聚合酶都在核内。DNA聚合酶γ在线粒体中，用于复制线粒体DNA。Leadingand

laggingstrands前导链和滞后链Thebasicschemeofreplicationofdouble-strandedchromosomalDNAin

eukaryotesfollowsthatforbacterialDNAreplication(seeTopicF3);aleading

strandandalaggingstrandaresynthesized,thelatterinvolvingdiscontinuous

synthesisviaOkazakifragments.TheRNAprimersrequiredaremadebyDNA

polymeraseαwhichcarriesaprimasesubunit.DNApolymeraseαinitiates

synthesisofthelaggingstrand,makingfirsttheRNAprimerandthen

extendingitwithashortregionofDNA.DNApolymeraseδthensynthesizes

therestoftheOkazakifragment.TheleadingstrandissynthesizedbyDNAδpolymerase.Theδenzymehas3’→5’exonucleaseactivityandsocanproof-readtheDNAmade,butDNApolymeraseαhasnosuchactivity.

真核生物双链染色体DNA复制的基本策略与细菌DNA复制相同（参看F3):也是合成前导链和滞后链，后者通过冈崎片段不连续合成。然而，在真核生物中，复制叉移动速率比原核生物中慢（约1/10)，并且两条新链由不同的DNA聚合酶合成：DNA聚合酶a通过冈崎片段合成滞后链，DNA聚合酶S合成前导链。RNA引物由携带引物酶亚基的DNA聚合酶a合成。S酶有3'—5'外切酶活性，可以校对合成的DNADNA聚合酶a没有这种活性，所以新生成的滞后链DNA可能是由另外的辅助蛋白质进行校对。Telomere

replication端粒复制

ThereplicationofalinearDNAmoleculeinaeukaryoticchromosomecreatesa

problemthatdoesnotexistforthereplicationofbacterialcircularDNAmole-cules.ThenormalmechanismofDNAsynthesis(seeabove)meansthatthe3’

endofthelaggingstrandisnotreplicated.Thiscreatesagapattheendofthe

chromosomeandthereforeashorteningofthedouble-strandedreplicated

portion.TheeffectisthatthechromosomalDNAwouldbecomeshorterand

shorteraftereachreplication.Variousmechanismshaveevolvedtosolvethis

problem.Inmanyorganismsthesolutionistouseanenzymecalledtelomerase

toreplicatethechromosomeends(telomeres).

真核生物染色体线性DNA分子的复制产生了一个在细菌环形DNA分子复制不存在的问题，按DNA合成的正常机制（见上文），滞后链3'端不被复制，这会造成染色体末端的空隙，因而缩短了被复制的双链部分，后果是每次复制会使染色体DNA越来越短。解决这个问题已演化出各种不同的机制。在许多机体中，解决的途径是用端粒酶来复制染色体的末端(端粒)。Telomere

replication端粒复制

Eachtelomerecontainsmanycopiesofarepeatedhexanucleotidesequencethat

isG-rich;inTetrahymenaitisGGGTTG.Telomerasecarries,asanintegralpartof

itsstructure,anRNAmolecule,partofwhichiscomplementarytothis

G-richsequence.Theexactmechanismofactionoftelomeraseisnotclear;Fig.3

showsonepossiblemodel.TheRNAmoleculeoftelomeraseisenvisagedto

hydrogen-bondtothetelomereend.Then,usingtheRNAasatemplate,telom-

erasecopiestheRNAtemplate(hencethisenzymeisareversetranscriptase;see

TopicI4)andaddssixdeoxynucleotidestothetelomereDNAend.Telomerase

thendissociatesfromtheDNA,re-bindsatthenewtelomereendandrepeatsthe

extensionprocess.Itcandothishundredsoftimesbeforefinallydissociating.The

newlyextendedDNAstrandcanthenactasatemplatefornormalDNAreplica-tion(laggingstrandsynthesisbyDNApolymerase)toformdouble-strandedchromosomalDNA.Thetwoprocesses,oftheDNAendsshorteningthrough

normalreplicationandoflengtheningusingtelomerase,areveryroughlyin

balancesothateachchromosomestaysapproximatelythesamelength.Telomere

replication端粒复制

每个端粒都含有许多富含G的六核苷酸序列的重复拷贝；在四膜虫(Tetrahymena)中是GGGTTG。端粒酶自身携带着作为其一部分结构的与这段富含G的序列互补的短RNA分子。端粒酶I作用的精确机制尚不清楚，图F4.3是一个假设模型。端粒酶中的RNA分子与端粒末端形成氢键。然后，以该RNA作为模板，端粒酶复制这个RNA模板（因此此酶是反转录酶（reversetranscriptase;参看14)并加入6个脱氧核苷酸到端粒DNA末端。然后端粒酶从DNA上脱落下来，再结合到新的端粒末端，重复延伸过程。它在最终脱落下来前可以重复