分子生物學(xué)第四章DNA的復(fù)制電子教案

1、第四章DNA的復(fù)制第四章 DNA 復(fù)制(DNA Replication) 1主要內(nèi)容1）DNA復(fù)制概覽2）細(xì)菌DNA復(fù)制3）真核DNA復(fù)制DNA的復(fù)制特點(diǎn)DNA復(fù)制的酶系統(tǒng)2 教學(xué)要求1）掌握原核生物和真核生物2）熟悉原核生物和真核生物 第一節(jié)DNA復(fù)制概述第二節(jié)DNA復(fù)制的酶學(xué) 第三節(jié)原核生物DNA復(fù)制 第四節(jié)真核生物DNA復(fù)制 第一節(jié) DNA 復(fù)制概述 （DNA Replication: An Overview）一、基本概念二、DNA的半保留復(fù)制三、復(fù)制起點(diǎn)、方式和方向四、DNA復(fù)制的半不連續(xù)性一、基本概念復(fù)制子(Replicon，復(fù)制單位或復(fù)制元):DNA中含有一定復(fù)制起點(diǎn)和復(fù)制終點(diǎn)的復(fù)

2、制單位。1.3萬一90萬不等A unit of the genome in which DNA contain a regi on from origi n to termi nator復(fù)制體(Replisome):復(fù)制叉處的許多酶和蛋白組成的復(fù)合體，協(xié)同動(dòng)作合成DNA。:The multi :- protein (30 ) ructure that assembles at replicating fork to undertake synthesis of DNA二、DNA的半保留復(fù)制(Semi-C on servatio n Replicati on)CsCl (Cesium chlor

3、ide) density gradient ultracentrifugation( CsCl 密度梯度超離心)Labeled E.Coli DNA with 15N14N :-三、復(fù)制起點(diǎn)、方向和方式?All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circles and comprise sin gle replicati ons.(單復(fù)制子)1、復(fù)制起點(diǎn)(origin，ori或O,復(fù)制原點(diǎn))復(fù)制開始處DNA分子的特定位置原核生物(Prokaryote):單復(fù)制起點(diǎn)，即整個(gè)染色體只

4、有一個(gè)復(fù)制單位真核生物(Eukaryote):多復(fù)制起點(diǎn)，即一個(gè)genome中有多個(gè)復(fù)制單位2、復(fù)制方向（復(fù)制過程的順序性）復(fù)制叉（Replication fork）:染色體中參與復(fù)制的活性區(qū)域，即復(fù)制正在發(fā)生的位點(diǎn)復(fù)制眼（replication eye）:電子顯微鏡下觀察正在復(fù)制的DNA，復(fù)制的區(qū)域形如一只眼睛真核生物的多復(fù)制子多個(gè)復(fù)制眼（1）單雙向復(fù)制取決于起點(diǎn)處有一個(gè)還是兩個(gè)復(fù)制叉（2）復(fù)制的多模式單起點(diǎn)、單方向（原核）多起點(diǎn)、單方向（真核）單起點(diǎn)、雙方向（原核）多起點(diǎn)、雙方向（真核）3、復(fù)制方式(1) 從新起始(de novo ini tiation )或復(fù)制叉式(replicatio

5、 n fork )(2) 置換式(Displacement form )又稱 D環(huán)復(fù)制(3) 共價(jià)延伸方式( covale nee elon gati on )或滾環(huán)式復(fù)制( rolli ng circle replicati on )DNA復(fù)制方式(1) 從新起始(de novo initiation)或復(fù)制叉式(replication fork )或 0 復(fù)制A replicati on eye forms a theta structure in circular DNA. :(2) 置換式 Displacementform，又稱 D 環(huán)復(fù)制線粒體和葉綠體 DNA的復(fù)制方式(3) 共價(jià)延

6、伸方式( covale nee elon gati on )或滾環(huán)式復(fù)制( rolli ng circle replicati on )由于復(fù)制時(shí)產(chǎn)生的滾環(huán)結(jié)構(gòu)形狀象d,又稱6復(fù)制病毒、細(xì)菌因子四、DNA復(fù)制的半不連續(xù)性(Semi-Disc on ti nu ous Replicatio n)復(fù)制方向的問題：岡崎片段(Okazaki fragment )1968 年岡崎(Reiji Okazaki )設(shè)計(jì)了兩組 實(shí)驗(yàn)，其一是 脈沖標(biāo) 記實(shí)驗(yàn)(pilse labeling experime nt )。岡崎利用T4噬菌體侵染E.cili(t-)菌株，并分別用dTTP(3H-T)進(jìn)行2, 7 ,15

7、 ,30 的脈沖標(biāo)記12分離提取T4噬菌體DNA，變性后，進(jìn)行 Cscl密度梯度離心，以檢測具放射性的沉降片斷，判斷片斷大小。結(jié)果表明經(jīng)不同標(biāo)記時(shí)間的，被3H-T標(biāo)記的新合成的DNA片段幾乎都為10-20S，即均為1000-2000 核苷酸大小(圖 3-)。第二組實(shí)驗(yàn)是脈沖追蹤實(shí)驗(yàn)(pulse chase experiment ),為了研究在脈沖標(biāo)記實(shí)驗(yàn)中所發(fā)現(xiàn)的10 20s的小片段在復(fù)制全過程中的發(fā)展結(jié)局，岡崎將實(shí)驗(yàn)菌株先進(jìn)行同位素標(biāo)記培養(yǎng)30秒，然后轉(zhuǎn)入正常培養(yǎng)基繼續(xù)培養(yǎng)數(shù)分鐘，分離DNA進(jìn)行密度梯度離心，發(fā)現(xiàn)小片段已被連接成為70 120S的大片段。為此將 DNA的這種復(fù)制方式稱為不連續(xù)

8、復(fù)制模式，將最初合成的10 20s片段稱為岡崎片段(圖 3 ) o如果兩條極性單鏈的DNA復(fù)制均按這種不連續(xù)的模式進(jìn)行，后隨鏈的合成可以在模板單鏈暴露到1000 2000核苷酸長度后，開始從5t 3合成岡崎片段，而先導(dǎo)鏈的合成從進(jìn)化的原則講，大可不必按岡崎片段的模式不斷地啟動(dòng)小片段的合成，既耗時(shí)又費(fèi)能。換言之，DNA的復(fù)制應(yīng)該是按一種半不連續(xù)的方式進(jìn)行，即先導(dǎo)鏈以連續(xù)復(fù)制的方式完成子代DNA的合成，而后隨鏈以不連續(xù)復(fù)制的方式完成岡崎片段的合成。但在Okazaki的脈沖標(biāo)記實(shí)驗(yàn)結(jié)果中，被標(biāo)記的DNA全部為小片段。其實(shí)在E.coli的細(xì)胞內(nèi)存在 dUTP和dTTP兩種類似物，其比例大約為1 : 3

9、00，而DNA聚合酶III 又不能區(qū)分這兩者，一旦將dUMP聚合到DNA分子中，必然會導(dǎo)致生物的基因表達(dá)與進(jìn)化過程中的潛在危險(xiǎn)，實(shí)際上 E.coli依靠了兩種機(jī)制阻止了dUMP摻入到DNA分子的過程。由 dut基因編碼的dUTP酶(dUTPase )能有效地將 dUTP , dUDP分解為dUMP,從而避免了 dUMP作為底物而 摻入到DNA分子中。即使 dUTPase能將絕大部分的 dUTP降解，但仍有約 1 / 1200的幾率dUTP 分子的逃逸，細(xì)胞內(nèi)的ung基因編碼的尿嘧啶一 N 糖苷酶(ungase )可以迅速地將已經(jīng)摻入到DNA分子中的dUMP切除，形成一個(gè)無嘌呤或嘧啶的Ap位點(diǎn)(

10、apurinic或apyrimidinic ),再由Ap內(nèi)切酶進(jìn)一步將 Ap位點(diǎn)酶解成缺口，以與其對應(yīng)的親代鏈為模板重新聚合和連接，完成切補(bǔ)修復(fù)過程顯然在先導(dǎo)鏈合成的初期過程中，約1200個(gè)核苷酸就有一個(gè)dUMP被切除的可能，在 Ap內(nèi)切酶未完全作用前提取DNA并進(jìn)行變性分析，就會得到與岡崎片段相似大小的1000 2000核苷酸的片段，這種片段也被稱為dUMP片段，Okazaki對dut 和ung 兩種突變體的研究結(jié)果證實(shí)。在dut突變體的脈沖標(biāo)記實(shí)驗(yàn)中，由于dUMP摻入的幾率增加，岡崎片段變短。而在ung突變體的脈沖標(biāo)記實(shí)驗(yàn)中，由于已摻入的 dUMP被切除的幾率降低，岡崎片段變長。岡崎選用連

11、接酶突變體(lig-)進(jìn)行的脈沖追蹤實(shí)驗(yàn)中，先導(dǎo)鏈的dUMP片段均不能被連接成大片段，(圖3 )o進(jìn)一步證明了在脈沖標(biāo)記實(shí)驗(yàn)中后隨鏈的岡崎片段與先導(dǎo)鏈的dUMP片段均以相似大小表現(xiàn)在其研究結(jié)果中。1978年Olivera據(jù)此提出了 DNA復(fù)制的半不連續(xù)模式。第二節(jié) DNA 復(fù)制的酶學(xué)(Enzymologyof DNA Replication)復(fù)制是在酶催化下的核苷酸聚合過程，需要多種高分子物質(zhì)共同參與DNA復(fù)制的體系底物：dNTP(dATP dGTP dCTP dTTP)聚合酶(polymerase): DNA-pol 依賴 DNA 的 DNA 聚合酶(DDDP)模板(template):解開

12、成單鏈的 DNA母鏈引物(primer):寡核苷酸片段，提供3-OH末端，使dNTP聚合其它酶和蛋白質(zhì)因子：解鏈酶，解旋酶，單鏈結(jié)合蛋白，連接酶一、DNA聚合酶二、DNA 連接酶(DNA Ligase)三、與DNA幾何學(xué)性質(zhì)相關(guān)的酶一、DNA聚合酶催化dNTP聚合到核酸鏈上的酶。是DNA復(fù)制的主要酶。全稱叫依賴DNA的DNA聚合酶(DNA dependentDNA polymerase )或 DNA 指導(dǎo)的 DNA 聚合酶(DNA-directedDNA polymerase )，均縮寫為 DDDP。(一) DNA聚合酶催化的反應(yīng)5宀3的聚合活性5宀3外切酶活性3宀5外切酶活性1、5至3的聚合

13、活性催化四種dNTP以磷酸二酯鍵一個(gè)一個(gè)地接到DNA鏈上去(dNMP)n + dNTP 宀(dNMP)n+1 + PPi聚合反應(yīng)的特點(diǎn)：(1) 以單鏈DNA為模板(2) 以dNTP為原料(3) 弓I物或DNA鏈提供3-OH(4) 聚合方向?yàn)?宀32、3 5外切核酸酶活性校對功能(proofreading)3、5 3外切核酸酶活性(二) 原核生物的DNA聚合酶(E.coli )1957 Arthur Kornberg 首次發(fā)現(xiàn)In fact, there are 5 polymerases to date, although we will focus only on- 3DNA pol IDN

14、A pol n DNA pol 川、1. DNA Polymerase I2. DNA Polymerase n和 川DNA pol n： 5、t 3、聚合酶活性及3 t 5外切核酸酶活性。DNA pol川：k由10個(gè)亞基組成，分別為 a 、B、t & S B、k及。是原核生物體內(nèi)真正起 復(fù)制作用的酶。-a亞基：5 t 3聚合酶活性-&亞基：3 t 5外切酶,校對和編輯-0亞基為裝配所必須亞基決定了 Pol : - III全酶的持續(xù)合成能力3. 三種大腸桿菌 DNA聚合酶特性之比較三種大腸桿菌DNA聚合酶的主要功能DNApol I -主要功能是切除引物，填補(bǔ)岡崎片段產(chǎn)生的空隙及DNA損傷的修復(fù)

15、。：DNApol :- n -是主要的修復(fù)酶DNApol :- m -是主要的復(fù)制酶三種DNA聚合酶在決定 DNA合成方面的共同特性 三種酶都只有5 聚合酶的功能，而沒有3T聚合酶功能，說明 DNA鏈的延伸只能從 5向3端進(jìn)行。 他們都沒有直接起始合成DNA的能力，只能在引物存在下進(jìn)行鏈的延伸，因此，DNA的合成必須有引物引導(dǎo)才能進(jìn)行。 三種酶都有核酸外切酶的功能，可對合成過程中發(fā)生的錯(cuò)誤進(jìn)行校正，而保證DNA復(fù)制的高度準(zhǔn)確性。為什么子鏈DNA延伸方向只能是5 t3二、DNA 連接酶(DNA Ligase )催化單鏈DNA切口上5-P和3-0H形成磷酸二酯鍵三、與DNA幾何學(xué)性質(zhì)相關(guān)的酶(一)

16、 解螺旋酶(helicase)模板對復(fù)制的指導(dǎo)作用在于堿基的準(zhǔn)確配對，而堿基卻埋在雙螺旋的內(nèi)部。只有把DNA解開成單鏈，它才能起模板作用。解螺旋酶是最早發(fā)現(xiàn)的與復(fù)制有關(guān)的蛋白質(zhì)，當(dāng)時(shí)稱為rep蛋白。作用是利用ATP供能，解開DNA雙鏈。復(fù)制相關(guān)蛋白的基因：dna A、dna B、dna Cdna X相應(yīng)的表達(dá)產(chǎn)物蛋白質(zhì)：Dna A、Dna B、Dna C Dna XDna A :辨認(rèn)復(fù)制起始位點(diǎn)Dna B :解螺旋酶Dna C :輔助解螺旋酶使其在起始點(diǎn)上結(jié)合并打開雙鏈。(二) DNA 拓?fù)洚悩?gòu)酶(topoisomerase)拓?fù)洌菏侵肝矬w或圖像作彈性移位而又保持物體不變的性質(zhì)。拓?fù)洚悩?gòu)酶：是

17、一類可改變DNA拓?fù)湫再|(zhì)的酶。對 DNA分子的作用是既能水解、又能連接磷酸二酯鍵。可松弛DNA超螺旋，有利于 DNA解鏈。拓?fù)洚悩?gòu)酶I (topo I )一蛋白。在原核生物曾被稱為主要作用是切開 DNA雙鏈中的一股，使 DNA解鏈旋轉(zhuǎn)中不打結(jié)，DNA變?yōu)樗沙跔顟B(tài)再封閉切口。 反應(yīng)不需要ATP拓?fù)洚悩?gòu)酶II ( topo II )?在原核生物又叫旋轉(zhuǎn)酶(gyrase)。?能切斷DNA雙鏈，使螺旋松弛。在 ATP參與下，松弛的 DNA進(jìn)入負(fù)超螺旋，再連接斷端。TopoI和TopoII松弛DNA負(fù)超螺旋(三) 單鏈DNA結(jié)合蛋白(SSB):在大腸桿菌，它是由 177個(gè)氨基酸殘基組成的同四聚體，結(jié)合單

18、鏈DNA的跨度約32個(gè)核苷酸單位。SSB與解開的DNA單鏈緊密結(jié)合， 防止重新形成雙鏈，維持模板處于單鏈狀態(tài)； 免受核酸酶降解。表4參與DNA復(fù)制的酶及蛋白質(zhì)酶或蛋白質(zhì)主要作用拓?fù)洚悩?gòu)酶類克服解鏈時(shí)打結(jié)及纏繞、松馳或引進(jìn)負(fù)超螺旋解鏈酶類解開DNA雙鏈單鏈DNA結(jié)合蛋白 維持已解開單鏈 DNA的穩(wěn)定引物酶合成RNA引物DNA聚合酶m DNA復(fù)制DNA聚合酶I水解引物、填補(bǔ)空隙、修復(fù)作用DNA 連接酶 催化雙鏈 DNA 中單鏈缺口的連接 小結(jié)1、基本概念DNA 復(fù)制 復(fù)制子 復(fù)制體 復(fù)制時(shí)期2 、半保留復(fù)制3 、復(fù)制起點(diǎn)：結(jié)構(gòu)特征復(fù)制方向：復(fù)制叉 復(fù)制眼 單雙向復(fù)制的決定條件 復(fù)制的多模式 復(fù)制方

19、式：復(fù)制叉式（從頭起始）D 環(huán)復(fù)制（置換式）b復(fù)制（滾環(huán)復(fù)制）4 、復(fù)制酶學(xué)1） 三種DNApol DNApol I和 山的主要活性和功能聚合活性外切活性（兩種）延伸方向2）DNA 連接酶3）和 DNA 的幾何學(xué)性質(zhì)相關(guān)的酶螺旋酶 旋轉(zhuǎn)酶5、 DNA 復(fù)制的半不連續(xù)性實(shí)驗(yàn)證據(jù) 先導(dǎo)鏈 后隨鏈4.3 Bacterial DNA replication4.3.1 Initiation （起始）4.3.2 Elongation （延伸）4.3.3 Termination of DNA replication4.3.1 Initiation （起始）?Study system: the E. coli

20、 origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes.?Initiation is divided into the following steps:1. recog niti on of the origi n（識另 U原點(diǎn)）2. separati on of pare ntal stra nds and stabilizati on of sin gle stra nds（分開雙鏈，穩(wěn)定單鏈）-3. i nitiation of daughter stra nd s

21、yn thesis through actio n of the PRIMOSOME（通過引發(fā)體起動(dòng)子代鏈的合成反應(yīng)）How do we know that there is only one origin of replication in E. coli?假定每個(gè)染色體都在同樣的一個(gè)起始點(diǎn)（ i）開始復(fù)制，那么距 i近的基因?qū)⑾缺粡?fù)制而復(fù)制得多 些，遠(yuǎn)離 i 的基因?qū)?fù)制得少些。因此在一個(gè)群體中離 i 點(diǎn)越近的基因出現(xiàn)頻率越高。? 假如復(fù)制是-單向的，基因頻率呈單向梯度-雙向的，基因頻率以 i 為中心呈雙向梯度How do we know that there is only one or

22、igin of replication in E. coli? 測定了大腸桿菌染色體上很多基因的頻率，發(fā)現(xiàn)以 OriC 基因附近為中心呈雙向下降。What does this experiment show?There is a single place on the chromosome where replication starts?Replication proceeds in both directions?The two replication forks meet at near 31 on the genetic mapThe structure of OriCOriCHow

23、is replication initiated?DnaA protein binds to 9-mers （9nt 聚合體 ）?DnaA together with HU 類組蛋白 denature the DNA?DnaB helicase 解旋酶 binds the open DNAInitiation of DNA replication in E. coli1. i nitial complex(起始復(fù)合體)?Al ong with the HU prote in, the dnaA protei n-ATP complex binds to the DNA, en compass

24、ing包圍the four nin e-mers .In all, this complex covers about 200bp.(隨同HU蛋白一起， dnaA protein-ATP 復(fù)合體結(jié)合到 DNA上，包圍4個(gè)9-mers區(qū)，總的覆蓋 200bp )2. Open complex(開放復(fù)合體)和 Prepriming complex(前引發(fā)復(fù)合體)DnaA蛋白使13bp重復(fù)單位熔解而形成開放性復(fù)合體，這一過程需要ATP。這時(shí)DnaB，和DnaC蛋白進(jìn)入DNA的熔解區(qū)與OriC結(jié)合形成前引發(fā)復(fù)合體。Separated strands in the oriC region are pre

25、vented from reannealing by the binding of single- stra nded binding prote in (SSB prote in).3. The primosome complex forms?A helicase (Dn aB) beg ins to unwind the stra nds.?SSB binds to preve nt reann eali ng.?Gyrase relieves the ten sio n.旋轉(zhuǎn)酶解除鏈的張力?Primase 引物酶 synthesizes a short strand of RNA.?Pr

26、imase binds to dnaB protein at oriC and forms a primosome引發(fā)體.Let s review the roles of the major players?Dn aA-Recog ni zes orig in and den atures the duplex?Dn aB-Helicase that unwinds the DNA?Dn aC-Required for DnaB binding?HU-hist on e-like protein stimulates in itiatio n?Primase (Dn aG) Syn thes

27、izes RNA primers?SSB-Si ngle stra nded DNA bin di ng protein?RNA polymerase Facilitates 促進(jìn) DnaA activity?DNA gyrase Relieves torsional 扭轉(zhuǎn)的 strain?Dam methylase Methylates GATC seque nces at oriCReview Initiation of DNA replication in E. coli1) oriC contains four 9 bp binding sites for the in itiator

28、 prote in Dn aA. Syn thesis of DnaA is coupled 聯(lián)系 to growth rate so that initiation of replication is also coupled to growth rate.2) DnaA forms a complex of 30-40 molecules, facilitating促進(jìn) melting 解鏈 of three 13 bp AT-richrepeat seque nee for DnaB binding.3) DnaB is a helicase that use the energy of

29、 ATP hydrolysis 水解 to further melt the double- stran ded DNA .4) SSB (sin gle-stra nded binding prote in) coats the unwin ded DNA to preve nt DNA ren aturi ng.5) DNA primase load to 負(fù)擔(dān) synthesizes a short RNA primer for synthesis of the leading strand.4. Primosome (弓丨發(fā)體):DnaB helicase and DNA primas

30、e4.3.2 Elo ngati on (延伸)?Elo ngation in volves ano ther complex of protei ns called the REPLISOME (復(fù)制體，復(fù)制顆粒 ).It isassembled from its comp onents each time replicati on occurs.E. coli replicati on machi nery(El on gatio n phase)1) Helicase-unwind DNA at replication fork in a reaction coupled to ATP

31、hydrolysis2) Single-strandedDNA binding proteins (ssb)-bind and stabilize the DNA in a single-strandedcon formatio n after melt ing by helicases3) Primosome-s yn thesizes RNA primers on laggi ng stra nd4) DNA Pol III-the true E. coli replicase5) DNA Topoisomerase II-relaxes supercoiled DNA that form

32、s ahead of replicati on fork-decate nates the fully replicated DNA6) DNA Pol Ireplaces RNA primers with DNA by nick-translation7) DNA ligase 連接酶 joins Okazaki fragmentsConcurrent 協(xié)同的 DNA Synthesis- Both strands replicate simultaneously at the same replication fork- The lagging template strandis loop

33、ed to invert the physical direction of synthesis, but not thechemical direction- DNA polymerase III functions as a dimer, with each core enzyme achieving synthesis on one or the other strand?If the polymerase complex is moving continuously along the leading strand, how does it discontinuously synthe

34、size DNA along the lagging strand in the opposite direction?4.3.3 Termination of DNA replication?Specific termination sites of DNA replication exist in E. coli.?Termination involves the binding of the tus gene product (tus protein) -ter binding protein, an inhibitor of the DnaB helicase (終止位點(diǎn)結(jié)合蛋白，是

35、DnaB 解旋酶的抑制劑)?This ter binding protein may act to prevent helicase from unwinding DNA (will therefore halt停止pol III and pol I action).?DNA replication produces two interlocking rings(連環(huán)) which must be separated.?This is accomplished via the enzyme topoisomerase. Termination of E. coli DNA replicatio

36、n Terminator sequences (終止序列) ： ?Trap the replication forks (使復(fù)制叉受到限制) ?Contain sequences that facilitate decate-nation and partitioning of daughter chromosomes(包含促進(jìn)連鎖分開和姐妹染色體分開的序列) .?300 bp? 需要 TUS ( Terminator Utilization Substance ) 識別終止序列Summary of steps in E. coli DNA synthesis:1. dnaA protein

37、melts duplex in oriC region.2. dnaB (helicase), along with dnaC and ATP binds to replication fork (dnaC protein exits). (Prepriming complex)3. Single strand binding protein (ssb protein) binds to separated strands of DNA and prevents reannealing.4. Primase complexes with helicase, creates RNA primer

38、s (pppAC(N)7-10) on the strands of the open duplex2 (Primase+helicase constitute the Primosome).5. After making the RNA primers, DNA pol III holoenzyme comes in and extends the RNA primer (laying down dNTPs) on the leading strand.4. As the replication fork opens up (via helicase + ATP action) leadin

39、g strand synthesis is an uninterrupted 不間斷的 process, the lagging strand experiences a gap.7. The gap region of the lagging strand can wind 旋緊 around one active site unit of the Pol III complex, and bound 阻止 Primase initiates an RNA primer in the gap region.8. On the lagging strand, Pol III extends t

40、he RNA primer with dNTP s as the lagging template strand is looped through the Pol III complex.9. After synthesis of a nascent 初始 fragment the lagging strand loop is released and the single strand region further up near the replication fork is subsequently looped through the Pol III complex.10. Step

41、s 7-9 are repeated.11. Meanwhile 其間 , Pol I removes the RNA primer regions of the Okazaki fragments via 5 to 3 exonuclease activity ( nick translation Pol I exits and ligase joints the DNA fragments (on lagging strand).4.4 Eukaryotic DNA replicati on441 Experimental system4.4.2 cell cycle4.4.3 ini t

42、iati on4.4.4 replicatio n forks4.4.5 nu clear matrix4.4.6 telomere replicati on4.4.1 Experimental systems?Yeast （Saccharomyces cerevisiae）酵母：has much smaller genome（1.4 X 107 bp in 16 chromosomes） and only 400 replic ons.?SV40 （simian virus 40猿猴病毒 40, 5 kb）: is good mammalian models for replication

43、fork.?Cell-free extract 無纟田胞提取物 prepared from Xenopus laevis （非洲爪蟾）eggs containing high concen tratio n of replicati on prote ins and can support in vitro replicati on.4.4.2細(xì)胞周期（cell cycle）的調(diào)控1. 細(xì)胞周期4個(gè)時(shí)期2. 檢驗(yàn)點(diǎn)及其調(diào)控3. 細(xì)胞周期蛋白和依賴于細(xì)胞周期蛋白的激酶1. 細(xì)胞周期4個(gè)時(shí)期?cell cycle細(xì)胞從一次有絲分裂結(jié)束到下一次有絲分裂完成所經(jīng)歷的一個(gè)有序過程。?細(xì)胞周期時(shí)相組成：?

44、間期（interphase）: G1 phase，S phase， G2 phase，細(xì)胞可由 G1期進(jìn)入一個(gè)非增殖期G0期或稱靜止期。?M phase:有絲分裂期（Mitosis）,胞質(zhì)分裂期（Cytok in esis）2. 檢驗(yàn)點(diǎn)（Checkpoint ）及其調(diào)控?細(xì)胞周期檢驗(yàn)點(diǎn)是細(xì)胞周期調(diào)控的一種機(jī)制。?細(xì)胞周期隨細(xì)胞周圍的環(huán)境而調(diào)整，以免受損傷的細(xì)胞發(fā)生增殖。?檢驗(yàn)點(diǎn)是當(dāng)外界條件不適合細(xì)胞分裂時(shí)，可以中斷細(xì)胞周期的一些階段。?主要檢驗(yàn)點(diǎn)位于 G1和G2的后期，在 G1期的R點(diǎn)，當(dāng)缺乏促細(xì)胞分裂原時(shí)，細(xì)胞會脫離細(xì)胞周期進(jìn)入非增殖的G0期。3. 細(xì)胞周期蛋白和依賴細(xì)胞周期蛋白的激酶細(xì)胞周

45、期是通過多種蛋白激酶復(fù)合物催化的蛋白磷酸化來實(shí)施調(diào)控的，這些復(fù)合物包含：regulatory sub un its （調(diào)節(jié)亞基）Cyclin （細(xì)胞周期蛋白）catalytic sub un it（催化亞基）Cycli n-depe nde nt kin ase（CDK）（依賴細(xì)胞周期蛋白的激酶）不同的復(fù)合物調(diào)控細(xì)胞周期中的不同時(shí)期。圖 Cyclin-dependent kinase （Cdk）4.4.3 Origins and InitiationOrigins and 人 Initiation （起始點(diǎn)與起始）ARS （自主復(fù)制序列）Lice nsing factor con trols

46、eukaryotic replicati on （特許因子）Differences in DNA betwee n eukaryotes and prokaryotesEukaryotic geno mes are much bigger Replication in eukaryotes occurs during a small portion of the cell cycleDNA in 九 eukaryotes is bound by hist onesEukaryotic chromosomes are lin ear圖 Multiple Replication OriginsOr

47、igins and Initiation（起始點(diǎn)與起始）?O nly once in one cell cycle（每個(gè)細(xì)胞周期只有一次復(fù)制）?Clusters of about 20-50 replicons （ 復(fù)制子）initiate simultaneously at defined times throughout S-phase （20-50個(gè)復(fù)制子在S期同時(shí)起始復(fù)制）?ln itiati on of each replic on within an in itiati on zone may occur at random（復(fù)制子可能在起始區(qū)域內(nèi)的任意位置起始）不同區(qū)域的染色質(zhì)起

48、始復(fù)制的時(shí)間不同?Early S-phase: euchromatin （ 常染色質(zhì)）replication?Late S-phase: heterochromatin （異染色質(zhì)）replication?Centromeric （著絲粒的）and telomeric （端粒的）DNA replicate at lastOrigins （起始點(diǎn)）Yeast orig in: the mini mal 11 bp consen sus:A/TTTTATA/GTTTA/TBound by origin recognition complex （ORC 起始識別復(fù)合體）activated 激活 by

49、 CDK.ARS: Auto no mously Replicati ng Seque nee （自主復(fù)制序列）。單細(xì)胞酵母的復(fù)制起點(diǎn)克隆進(jìn)原核生物的質(zhì)粒，由于這些復(fù)制起點(diǎn)序列允許質(zhì)粒在酵母中復(fù)制而被稱之為ARS。The yeast ARSLicensing factor controls eukaryotic replication（特許因子控制真核生物DNA 復(fù)制）Lice nsing factor con trols eukaryotic rereplicati on DNA Replicati on?The mach inery of replicatio n-Additional f

50、eatures of eurkaryotic cells?One replicati on per cycle- con trol?The orig in of replicati on passage through a series of steps? Origin of replication bound by ORCs? Lice nsing factors bi nd assemble the prereplicatio n complex? Lice nsing factors -at least six Mcm2-Mcm7? Mcm prote ins move with the

51、 replicatio n fork? Mcm proteins are then displaced from DNA but remain in nucleus? Mcm proteins cannot reassociate with an origin of replication which has already fired 444 replicat on fork?The mach inery of replicatio n?Additional features of eurkaryotic cells-Chromati n structure?Nucleosomes and

52、the replicati on fork-Hist ones - H3H4 tetramers 四聚體 remai n in tact and are distributed betwee n the daughter duplexes-Old and new H3H4 tetramers found on each duplex-H2A/H2B dimers -separate and bind ran domly to H3H4 tetramers already in placeReplicati on of NucleosomesEukaryotic DNA is packaged

53、with hist ones in structures called n ueleosomes.Whathappensto the nueleosome when the replication fork and thereplicationmach inery pass by and ope n up the DNA double stra nd?Nucleosomes are found properly - spaced on both postreplicative DNA strands immediately after passage of replicati on fork.圖 A model for nucleosome replication圖 A model for nu