1、 DNA DNA的复制的复制God said to them:“Be fruitful and multiply！”圣经创世纪第一节第一节 DNADNA复制概貌复制概貌一、DNA复制的半保留性二、复制过程中的顺序性复制从原点开始边解链边复制w 为什么不采取先将DNA全部解链，然后再分别进行复制的方法？三、DNA的半不连续复制冈崎片段的发现DNA只能从5到3方向合成不能从两端同时复制3，5磷酸二酯键的形成第二节 DNA的复制酶和相关蛋白一、DNA聚合酶w种类：DNA聚合酶I：1956年，Kornbergw具有聚合酶、3-5 外切核酸酶、5-3 外切核酸酶活性w主要生理功能：主要的DNA修复酶，并
2、参与半保留复制Roger,Kenneth,Sylvy,Arthur and Thomas.DNA聚合酶II：没有5-3 外切核酸酶活性，次要的DNA修复酶DNA聚合酶III：是真正的DNA复制酶w多亚基w多层次组装DNA聚合酶IV和V：参与SOS修复wDNA聚合酶III的结构：DNA聚合酶 3-5核酸外切酶 激活外切酶活性 使核心酶结合为二聚体；结合复合物 结合ATP 与亚基结合与和亚基结合 与SSB 蛋白结合 与和亚基结合 sliding clamp（滑动夹环）核心酶 Pol IIIPol IIIPol III*sliding clamp二、解螺旋酶(helicase)：A helicase
3、 is an enzyme that uses energy provided by NTP hydrolysis to separate the strands of a nucleic acid duplex 三、SSB蛋白（单链结合蛋白）The single-strand binding protein(SSB)attaches to single-stranded DNA,thereby preventing the DNA from forming a duplex 防止单链复性维持单链刚性状态避免单链降解The SSB binds as a monomer,but typicall
4、y in a cooperative manner in which the binding of one protein molecule makes it much easier for another to bind 四、引发酶(primase):The primase is a type of RNA polymerase that synthesizes short segments of RNA that will be used as primers for DNA replication.为什么DNA的合成需要RNA引物？五、旋转酶(gyrase)：a Type II topo
5、isomerase,acts to overcome the torsional stress imposed upon unwinding by introducing negative supercoils at the expense of ATP hydrolysis.复制叉前进带来扭曲张力拓扑异构酶II：引入负超螺旋六、DNA连接酶（DNA ligases）DNA ligase makes a bond between an adjacent 3-OH and 5-phosphate end where there is a nick in one strand of duplex
6、DNA.1、酶活化2、AMP转移3、3-OH亲核攻击第三节 DNA的复制过程一、原核生物的复制（E.coli）（一）复制的起始：复制原点：Ori C，含两个系列的重复单位，3个13bp重复序列（富含AT），和4个9bp重复序列（识别位点）w起始过程：The origin is initially recognized by a protein that forms a large complex with DNA.A short region of AT-rich DNA is melted.Helicase is bound to the complex and creates the re
7、plication fork.The first nucleotides of the new chain are synthesized into the primer wDnaA protein is the initiation factor:wThe four 9 bp consensus sequences on the right side of oriC provide the initial binding sites for DnaA.It binds cooperatively to form a central core around which oriC DNA is
8、wrapped.wThen DnaA acts at three A-T-rich 13 bp tandem repeats located in the left side of oriC.In the presence of ATP,DnaA melts the DNA strands at each of these sites to form an open complex wHU protein has the capacity to bend DNA,and is involved in building the structure that leads to formation
9、of the open complex.wTranscriptional activation:RNA polymerase could be required to read into the origin from adjacent transcription units.The act of transcription could be associated with a structural change that assists melting of DNA.HuwThe replication fork is generated w2-4 monomers of DnaA bind
10、 at the origin,and they recruit 2 prepriming complexes of DnaB-DnaC to bind,so that there is one for each of the two(bidirectional)replication forks.wDnaB protein is delivered to oriC by DnaC protein in the form of a hexameric(DnaB:DnaC)6 complex,but DnaC protein does not enter the protein assemblag
11、e at oriC.wDnaB protein has helicase activity and it further unwinds the DNA in the pre-priming complex in both directions,assisted by DNA gyrase.SSB tetramers coat single-stranded regions as they arise.wPrimer synthesis:each DnaB activates a DnaG primase,in one case to initiate the leading strand,a
12、nd in the other to initiate the first Okazaki fragment of the lagging strand.（二）复制的延伸：1.复制体：酶和相关蛋白在复制叉形成的超分子复合物t 先导链和后随链的同时复制Lagging template strand通过复制体形成一个环状结构，primase已经合成一条引物2随着冈崎片段的合成，环逐渐变大，直到2片段末端接近前一冈崎片段的引物复制体释放环，primase合成一个新的引物环型DNA的复制类型：w型（E.Coli）:an origin creates two replication forks that
13、 move in opposite directions wD-loop(mammalian mitochondrial DNA)：mtDNA的复制在H链的复制起点OH开始合成;当置换链通过L链的复制起点OL时，开始合成新的H链;新的L链合成完成，子代双链被释放。Key concepts:Mitochondria use different origin sequences w Rolling circles（X174 phage）wA protein nicks the origin and binds to 5 end.wUsing the rolling circle,the 3 OH
14、end of the nick is extended into a new chain.wThe positive strand has grown to double length and replication fork is back at the orgin.wA protein cuts the bouble-length positive strand in half,releasing the displaced half and binds to the new 5 end.wReleasd plus strand forms covalent circle.w Restar
15、t of stalled replication forks:wInitiation of X174 replication requires the primosome complex to displace SSB from the origin.The primosome consists of six proteins:PriA,PriB,PriC,DnaT,DnaB,and DnaC.A primosome assembles at a unique site,called the assembly site(pas).wThe importance of the primosome
16、 for the bacterial cell is that it is used to restart replication at forks that stall when they encounter damaged DNA.At forks without a lagging strand,the single-stranded lagging-strand template can be bound directly by DnaB.At forks with a lagging strand,this strand must be unwound by PriA helicas
17、e to generate a single-stranded binding site for DnaB loading（三）、复制的终止：n环形DNA：n单向复制：复制终点复制起点n双向复制：两个生长点的简单碰撞 (无固定 终点)有固定终点（如E.Coli）E.coli的Ter终止位点 当复制叉前移，遇到20bp重复性终止子序列（Ter）时，Ter-Tus复合物能阻挡复制叉的继续前移，等到相反方向的复制叉到达后在DNA拓扑异构酶IV的作用下使复制叉解体，释放子链DNA wT7 DNA两端各有一段重复的数百个核苷酸，叫末端冗余。w两个5端有空缺的分子通过3端凸出的重复序列互补配对。w然后DN
18、A聚合酶从3端延伸填补起这个空缺，留下最后的裂隙由连接酶封闭，产生了一个连环分子。w再由特异性限制内切酶在连接处交错切割，产生3 凹端w由DNA多聚酶在3-OH上延伸填满新的空缺，产生两条完整的双链。2.线性DNA：环化，如 DNA 形成连环分子，如T7 DNAw真核生物复制特点：复制速度慢基因组较大，有多个复制起点在全部复制完成以前，起点不开始新一轮复制二、真核生物的复制w真核生物DNA聚合酶：表表2-11 真核生物真核生物DNA聚合酶的特性比较聚合酶的特性比较性质性质DNA聚合酶聚合酶DNA聚合酶聚合酶DNA聚合酶聚合酶DNA聚合酶聚合酶DNA聚合酶聚合酶亚基数4122-31在细胞内分布核
19、内核内线粒体核内核内(?)功能DNA引物合成损伤修复线粒体DNA复制主要DNA复制酶DNA复制(?)35外切无无有有有53外切无无无无无w复制过程中的核小体w复制叉前进是核小体解离为H32H42 四聚体和 H2AH2B 二聚体w新合成的组蛋白也被组装成H32H42 四聚体和 H2AH2B 二聚体w旧的和新的四聚体、二聚体，在CAF-1因子的帮助下，随机地被组装到复制叉后新形成的核小体中端粒的复制：端粒(Telomere)端粒是真核染色体的末端序列，富含G。主要由一串十分简单和串联重复的序列组成如四膜虫：GGGGTT端粒功能：操持染色体的稳定性端粒酶(Telomerase)：一种含RNA的蛋白复
20、合物，能使端粒延伸。酶所含的RNA长约150bp，是合成端粒的模板。端粒酶实际上是一种逆转录酶。复制过程：除去子代链5端引物2.在母链3末端添加端粒序列3.以新合成的端粒序列为模板合成DNA4.除去步骤3中所使用的引物，缺口被保留，但未丢失DNAElizabeth H.Blackburn Jack W.Szostak Carol W.Greider The Nobel Prize in Physiology or Medicine 2009for the discovery of how chromosomes are protected by telomeres and the enzyme
21、 telomerase三、复制的调控：w复制原点的甲基化：复制原点Ori C包含11个拷贝的GATC序列，该序列是DAM 甲基化酶的作用位点。DAM甲基化酶使腺嘌呤上的N6位点甲基化。复制前，双链上回文序列中的A都被甲基化。子代链复制过程中，产生半甲基化DNA。对dam-E.Coli 的研究表明，半甲基化的Ori C不能发动一轮新的复制在复制过程中，Ori C的半甲基化状态约保留13 min。而在基因组其它区域的GATC位点，在复制后1.5 min内即被甲基化。wColEl质粒DNA的复制调控：引物RNA前体的转录起始于复制起点上游555个核苷酸处，需经RNaseH加工后产生有555个核苷酸的引物，然后由DNA聚合酶I在引物的3末端起始DNA合成。RNA1的编码区在引物RNA编码区的5末端，转录方向与引物RNA相反，因此与引物RNA的5末端互补RNA1通过氢键配对与引物RNA前体相互作用，阻止了RNase H加工引物前体，使其不能转化为有活性的引物而对复制起负调控作用。w当不存在RNA 1时，RNA引物前体形成自身的发夹结构，起类似终止子的作用w当前体RNA与RNA1互补配对时，类终止子效应消失，引物前体的转录被继续，阻止了RNase H加工引物前体
