大學(xué) 生物化學(xué) F4

BIOCHEMISTRYF4DNAREPLICATIONINEUKARYOTES應(yīng)用化學(xué)1201張隴濤1215020129F4DNAREPLICATIONIN

EUKARYOTESIneukaryotes,thecellcycleconsistsofG1,S,G2andMphases.

Most

differencesinthecycletimesofdifferentcellsareduetodifferencesin

thelengthoftheG1phase.QuiescentcellsaresaidtobeintheG0phase.Cellcycle真核生物中，細(xì)胞周期包括Gi、S、和M期。不同細(xì)胞的不同周期時(shí)間主要是由Gi期的長(zhǎng)短決定。靜止細(xì)胞（quiescentcells)認(rèn)為是處于GQ期。細(xì)胞周期DNAreplicationoccursonlyintheSphase.Itoccursatmany

chromosomalorigins,isbi-directionalandsemi-conservative.Setsof

20–80repliconsactasreplicationunitsthatareactivatedinsequence.MultiplerepliconsDNA復(fù)制只發(fā)生于S期（Sphase)。它發(fā)生于染色體上許多起始點(diǎn)，是雙向而且是￥保留復(fù)制。20?80個(gè)成套的復(fù)制（repli-con)依次被激活作為復(fù)制單位。多復(fù)制子KeyNotesDNApolymerasesαandδreplicatechromosomalDNA,DNA

polymerasesβandεrepairDNA,andDNApolymeraseγreplicates

mitochondrialDNA.FiveDNApolymerasesDNA聚合酶α和δ復(fù)制染色體DNA，DNA聚合酶β和ε修復(fù)DNA,DNA聚合酶γ復(fù)制線(xiàn)粒體DNA。5種DNA聚合酶DNApolymeraseαandδsynthesizethelaggingstrand,viaOkazaki

fragments.TheRNAprimersaresynthesizedbyDNApolymeraseαwhichcarriesaprimasesubunit.DNApolymeraseδsynthesizesthe

leadingstrand.Leadingandlagging

strandsDNA聚合酶α將δ片段合成滯后鏈，而聚合酶δ合成前導(dǎo)鏈。RNA引物由DNA聚合酶α來(lái)合成，該酶帶有引物酶的一個(gè)亞基。前導(dǎo)鏈和滯后鏈Telomerase,aDNApolymerasethatcontainsanintegralRNAthatacts

asitsownprimer,isusedtoreplicateDNAattheendsofchromosomes

(telomeres).Telomerereplication端粒酶（telomerase)，一種自身含有可作為引物的內(nèi)在RNA的DNA聚合酶，用于在染色體末端合成DNA(端粒，telomeres)。端粒復(fù)制NucleosomesdonotdissociatefromtheDNAduringDNAreplication;

rathertheymustopenuptoallowthereplicationapparatustopass.Both

daughterDNAmoleculeshaveoldhistonesboundtothembutnew

histonesmustalsobesynthesizedtoallowalltheDNAtobepackaged

correctlyinnucleosomes.Replicationof

chromatinDNA復(fù)制時(shí)核小體不從DNA上解離，但它們必須開(kāi)放以允許復(fù)制體(replicationapparatus)通過(guò)。兩個(gè)子代DNA分子都與先前的組蛋白組合，但還必須合成新的組蛋白使所有DNA在核小體中能正確包裝。染色質(zhì)的復(fù)制Relatedtopics

DNAstructure(F1)

DNAreplicationinbacteria(F3)相關(guān)主題

DNA結(jié)構(gòu)（F1)

原核生物中DNA的復(fù)制(F3)Cellcycle

細(xì)胞周期Thelifeofaeukaryoticcellcanbede?nedasacellcycle(Fig.1).MitosisandcelldivisionoccurintheMphasewhichlastsforonlyabout1h.ThisisfollowedbytheG1phase(Gforgap),thentheSphase(Sforsynthesis),duringwhichtimethechromosomalDNAisreplicated,and?nallytheG2phaseinwhichthecells

prepareformitosis.Eukaryoticcellsinculturetypicallyhavecellcycletimesof

16–24hbutthecellcycletimecanbemuchlonger(>100days)forsomecellsin

amulticellularorganism.Mostofthevariationincellcycletimesoccursby

differencesinthelengthoftheG1phase.Somecellsinvivo,suchasneurons,

stopdividingcompletelyandaresaidtobequiescent,lockedinaG0phase.真核細(xì)胞的一生可以定義為一個(gè)細(xì)胞周期（cellcycle)(圖F4.1)。有絲分裂和細(xì)胞分裂發(fā)生在約持續(xù)1小時(shí)的M期，緊接著是（^期（G代表gap),然后是S期（S代表synthesis)，在此期間染色體DNA被復(fù)制，最后是細(xì)胞準(zhǔn)備有絲分裂的G2期。培養(yǎng)的真核細(xì)胞的周期時(shí)間通常為16~24小時(shí)，但在多細(xì)胞機(jī)體中一些細(xì)胞的周期時(shí)間可以很長(zhǎng)（>100天）。細(xì)胞周期長(zhǎng)度的變化大部分發(fā)生在(?期。一些體內(nèi)細(xì)胞，如神經(jīng)元，完全停止分裂，被認(rèn)為是靜止的，處于仏期（Gcphase)。Multiplereplicons多復(fù)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，染色體DNA的復(fù)制只發(fā)生在細(xì)胞周期的S期。與細(xì)菌DNA相似（參看F3)，真核生物的DNA也是半保留復(fù)制。染色體線(xiàn)性DNA分子的復(fù),制起始于多個(gè)起始點(diǎn)，隨種系與組織不同，大約每3?300kbDNA就有一個(gè)起始點(diǎn)，而且從每個(gè)起始點(diǎn)處雙向進(jìn)行。多起始點(diǎn)的應(yīng)用對(duì)于保證染色體DNA在必需時(shí)間內(nèi)完成復(fù)制是至關(guān)重要的。在每個(gè)起始點(diǎn)，復(fù)制泡由兩個(gè)反向運(yùn)動(dòng)的復(fù)制叉組成。由一個(gè)起始點(diǎn)控制的箄制DNA稱(chēng)為一個(gè)復(fù)制子。DNA合成不斷進(jìn)行直至復(fù)制泡融合在一起(圖F4.2)。Fig.1.

Theeukaryoticcellcycle.TheSphaseistypically6–8hlong,G2isaphaseinwhich

thecellpreparesformitosisandlastsfor2–6h,mitosisitself(M)isshortandtakesonlyabout

1h.ThelengthofG1isveryvariableanddependsonthecelltype.CellscanenterG0,

aquiescentphase,insteadofcontinuingwiththecellcycle.圖F4.1真核細(xì)胞周期。S期通常6~8小時(shí)；（?期是細(xì)胞準(zhǔn)備有絲分裂的時(shí)期，延續(xù)2~6小時(shí)；有絲分裂（M期）時(shí)間短，僅1小時(shí)左右；（?的長(zhǎng)短變化很大，依賴(lài)于細(xì)胞類(lèi)型。細(xì)胞亦可進(jìn)人Go期即靜止期.則不再進(jìn)人細(xì)胞周期Fig.2.

ReplicationofeukaryoticchromosomalDNA.Replicationbeginsatmanyoriginsand

proceedsbi-directionallyateachlocation.Eventuallythereplicationeyesmergetogetherto

producetwodaughterDNAmolecules,eachofwhichconsistsofoneparentalDNAstrand

(thinline)andonenewlysynthesizedDNAstrand(thickline).圖F4.2真核生物染色體DNA的復(fù)制。復(fù)制開(kāi)始于多個(gè)起始點(diǎn)并在'每個(gè)位點(diǎn)雙向進(jìn)行。最后這些復(fù)制眼融合產(chǎn)生兩個(gè)子代DNA分子，每個(gè)分子都含有一條來(lái)自母鏈的DNA鏈（細(xì)線(xiàn)）和一條新合成的DNA鏈（粗線(xiàn)）

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

unitsareactivatedinasetorderuntileventuallythewholechromosomehas

beenreplicated.Transcriptionally-activeDNAappearstobereplicatedearlyinSphase,whilstchromatinthatiscondensedandnottranscriptionallyactiveis

replicatedlater.Multiplereplicons多復(fù)制例子

染色體的各部分不是同時(shí)進(jìn)行復(fù)制的，而是在染色體的某一部分可以一存在很^復(fù)-制服，—而在另=■部分卻二■個(gè)也沒(méi)有。因此復(fù)制起始點(diǎn)是成簇溻活（activatedinclusters)，稱(chēng)為復(fù)制單位（replicationunits)，每個(gè)復(fù)制單位含有20?80個(gè)起始點(diǎn)。在S期不同的復(fù)制單位被依次激活，直到整條染色體完成復(fù)制。轉(zhuǎn)錄活躍的基因在S期較早被復(fù)制，而濃縮的轉(zhuǎn)錄不活躍的染色質(zhì)復(fù)制得較遲。Five

DNApolymerases5種DNa聚合酶Eukaryoticcellscontain?vedifferentDNApolymerases;α,β,γ,δandε.The

DNApolymerasesinvolvedinreplicationofchromosomalDNAareαandδ.DNApolymerasesβandεareinvolvedinDNArepair.Allofthesepolymerases

exceptDNApolymeraseγarelocatedinthenucleus;DNApolymeraseγis

foundinmitochondriaandreplicatesmitochondrialDNA.真核生物細(xì)胞含有5種不同的DNA聚合酶：α,β,γ,δ和ε。在染色體'DNA復(fù)制中所用的DNA聚合酶為α和δ。DNA聚合酶β和ε用于DNA修復(fù)。除了γ外所有的DNA聚合酶都在核內(nèi)。DNA聚合酶γ在線(xiàn)粒體中，用于復(fù)制線(xiàn)粒體DNA。Leadingand

laggingstrands前導(dǎo)鏈和滯后鏈Thebasicschemeofreplicationofdouble-strandedchromosomalDNAin

eukaryotesfollowsthatforbacterialDNAreplication(seeTopicF3);aleading

strandandalaggingstrandaresynthesized,thelatterinvolvingdiscontinuous

synthesisviaOkazakifragments.TheRNAprimersrequiredaremadebyDNA

polymeraseαwhichcarriesaprimasesubunit.DNApolymeraseαinitiates

synthesisofthelaggingstrand,making?rsttheRNAprimerandthen

extendingitwithashortregionofDNA.DNApolymeraseδthensynthesizes

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

真核生物雙鏈染色體DNA復(fù)制的基本策略與細(xì)菌DNA復(fù)制相同（參看F3):也是合成前導(dǎo)鏈和滯后鏈，后者通過(guò)岡崎片段不連續(xù)合成。然而，在真核生物中，復(fù)制叉移動(dòng)速率比原核生物中慢（約1/10)，并且兩條新鏈由不同的DNA聚合酶合成：DNA聚合酶a通過(guò)岡崎片段合成滯后鏈，DNA聚合酶S合成前導(dǎo)鏈。RNA引物由攜帶引物酶亞基的DNA聚合酶a合成。S酶有3'—5'外切酶活性，可以校對(duì)合成的DNADNA聚合酶a沒(méi)有這種活性，所以新生成的滯后鏈DNA可能是由另外的輔助蛋白質(zhì)進(jìn)行校對(duì)。Telomere

replication端粒復(fù)制

ThereplicationofalinearDNAmoleculeinaeukaryoticchromosomecreatesa

problemthatdoesnotexistforthereplicationofbacterialcircularDNAmole-cules.ThenormalmechanismofDNAsynthesis(seeabove)meansthatthe3’

endofthelaggingstrandisnotreplicated.Thiscreatesagapattheendofthe

chromosomeandthereforeashorteningofthedouble-strandedreplicated

portion.TheeffectisthatthechromosomalDNAwouldbecomeshorterand

shorteraftereachreplication.Variousmechanismshaveevolvedtosolvethis

problem.Inmanyorganismsthesolutionistouseanenzymecalledtelomerase

toreplicatethechromosomeends(telomeres).

真核生物染色體線(xiàn)性DNA分子的復(fù)制產(chǎn)生了一個(gè)在細(xì)菌環(huán)形DNA分子復(fù)制不存在的問(wèn)題，按DNA合成的正常機(jī)制（見(jiàn)上文），滯后鏈3'端不被復(fù)制，這會(huì)造成染色體末端的空隙，因而縮短了被復(fù)制的雙鏈部分，后果是每次復(fù)制會(huì)使染色體DNA越來(lái)越短。解決這個(gè)問(wèn)題已演化出各種不同的機(jī)制。在許多機(jī)體中，解決的途徑是用端粒酶來(lái)復(fù)制染色體的末端(端粒)。Telomere

replication端粒復(fù)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.Itcandothishundredsoftimesbefore?nallydissociating.The

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

normalreplicationandoflengtheningusingtelomerase,areveryroughlyin

balancesothateachchromosomestaysapproximatelythesamelength.Telomere

replication端粒復(fù)制

每個(gè)端粒都含有許多富含G的六核苷酸序列的重復(fù)拷貝；在四膜蟲(chóng)(Tetrahymena)中是GGGTTG。端粒酶自身攜帶著作為其一部分結(jié)構(gòu)的與這段富含G的序列互補(bǔ)的短RNA分子。端粒酶I作用的精確機(jī)制尚不清楚，圖F4.3是一個(gè)假設(shè)模型。端粒酶中的RNA分子與端粒末端形成氫鍵。然后，以該RNA作為模板，端粒酶復(fù)制這個(gè)RNA模板（因此此酶是反轉(zhuǎn)錄酶（reversetranscriptase;參看14)并加入6個(gè)脫氧核苷酸到端粒DNA末端。然后端粒酶從DNA上脫落下來(lái)，再結(jié)合到新的端粒末端，重復(fù)延伸過(guò)程。它在最終脫落下來(lái)前可以重復(fù)