第二章染色体与DNA(2)DNA复制,修复,转座(1)

1、原核生物DNA的复制In 1963, John Cairns Technique: Grew E. coli in 3H thymidine Waited till cells were in the middleof replication Lysed the cells very very gently Spread the lysate on an EM grid Exposed the grid to X-ray film forTWO months.Replication of the E. colichromosomeWhat Cairns experiment showed: E

2、. coli has a circular chromosome. E. coli has a single origin of replication. In E. coli, replication and unwinding aresimultaneous.What Cairns did not show: Is replication UNIdirectional or BIdirectional?Bidirectional Replication(双向复制) 无论是原核生物还是真核生物，DNA的复制主要是从固定的起始点以双向等速复制方式进行的。 复制叉以DNA分子上某一特定顺序为起点

3、，向两个方向等速生长前进。Bidirectional Replication(双向复制)起始点复制叉复制叉DNA Synthesis 由于DNA双螺旋的两条链是反向平行的，因此两个模板极性不同。 所有已知DNA聚合酶的合成方向都是53，为 了 解 释 DNA 的 等 速 复制现象 , 日本学者冈崎(Okazaki) 等提出了DNA 的半 不 连 续 复 制 模 型 (semi-discontinuous replication) .半不连续复制 Semi-discontinuous replication冈崎片段Okazaki fragmentLeading strandReplication

4、 forkLagging strand前导链的连续复制和滞后链的不连续复制在生物界是有普遍性的，因而称之为DNA的半不连续复制。1、用3H脱氧胸苷短时间标记后提取DNA，得到不少平均长度为2-3kb DNA片段。2、用DNA连接酶温度敏感突变株进行实验，在连接酶不起作用的温度下，有大量小片段累积，说明复制过程中至少有一条链首先合成较短的片段，然后再生成大分子DNA。3H Thymidine pulse-chase labeling and alkaline sucrosegradient: discovery of semi-discontinous replicationDNA的半不连续复制

5、（semi-discontinuous replication）The short discontinuous segments are calledOkazaki Fragments.In bacteria they are approximately 1000 nt inlength; in eukaryotes they are approximately200 nt in length. 亲代DNA分子为模板 四种脱氧三磷酸核苷(dNTP)为底物 提供3-OH末端的引物 多种酶及蛋白质DNA拓扑异构酶、DNA解链酶、单链结合蛋白、引物酶、 DNA聚合酶、RNA酶以及DNA连接酶等DNA

6、 复制的体系DNA复制的基本过程 复制的起始 (initiation) DNA链的延伸 (elongation) 复制的终止 (termination)1. DNA复制的起始 复制起始原点 DNA双螺旋的解旋 复制的引发大肠杆菌(E. coli)的 OriC 复制原点大肠杆菌基因组的复制原点位于天冬酰胺合酶和ATP合酶操纵子之间，全长245 bp, 称为oriC。参与DNA复制起始和引发的蛋白质 DNA解旋酶(DNA helicase)催化DNA双链的解链过程。 单链DNA结合蛋白(single strand DNA bindingprotein)：以四聚体形式存在于复制叉处，只保持单链的存在

7、，并不能起解链作用。 DNA拓扑异构酶(DNA topoisomerase)消除DNA双链的超螺旋堆积。 引物酶(primase)合成一小段RNA引物，为DNA新链的合成提供3-OH末端。DNA helicases separate the twostrands of the double helixDNA双螺旋的解旋Binding of SSB to DNA inhibits theformation of intramolecular base pairsDNA双螺旋的解旋Action of topoisomeraseat the replication forkTopoisomerase

8、 rapidlyremove the positivesupercoils accumulate infront of the replicationfork.DNA 聚 合 酶 只 能 延 长 已 存 在 的DNA链，而不能从头合成DNA链，那么，新DNA的复制是怎样开始的呢？DNA polymerase requires a 3-OH endto initiate replicationThe 3OH end is called a primer.A primer is a short sequence (often ofRNA) that is paired with one stran

9、d ofDNA and provides a free 3 -OH end atwhich a DNA polymerase starts synthesisof a deoxyribonucleotide chain.The primase is a type of RNA polymerasethat synthesizes short segments of RNAthat will be used as primers for DNAreplication.Substrates required for DNA synthesisBoth a primer and a template

10、 are essential for all DNA synthesis由大肠杆菌oriC复制起始点处引发的DNA复制过程大约20个DnaA蛋白在ATP的作用下与oriC处的4个9 bp保守序列相结合。在Hu蛋白和ATP的共同作用下，DnaA复制起始复合物使3x13 bp直接重复序列变形，形成开链。DnaB（解链酶）六体分别与单链DNA相结合（需要DnaC的帮助）进一步解开DNA双链。与引物结合，起始DNA复制。 DNA聚合酶 滑动夹（Sliding DNA clamp） RNA酶(RNase H 等)在复制完成后切除RNA引物。 DNA连接酶 (DNA ligase)通过生成35-磷酸二酯键

11、连接两条DNA链。2. DNA链的延伸DNA链的延伸需要的蛋白质:DNA polymerase use a single active siteto catalyze DNA synthesisDNA polymerase bound to a primer:template junctionpalmTwo metal ions bound to DNApolymerase catalyze nucleotide additionDNA template strandprimer stranddNTPActive site of a DNA polymerasepalmDNA polymera

12、sepalm domain:1. Contains two catalytic sites, onefor addition of dNTPs and one forremoval of the mispaired dNTP.2. The polymerization site bindsto two metal ions that alter thechemical environment around thecatalytic site and lead to thecatalysis. (how?)3. Monitors the accuracy of base-pairing for

13、the most recentlyadded nucleotides by formingextensive hydrogen bond contactswith minor groove of the newlysynthesized DNA.FingerDNA polymeraseFinger domain:Binds to the incomingdNTP, encloses thecorrect paired dNTP tothe position for catalysisBends the template toexpose the onlynucleotide at thetem

14、plate that ready forforming base pair withthe incoming nucleotideStabilization of thepyrophosphateDNA polymerase “grips” the template and theincoming nucleotide when a correct base pair is madeIn the fingerdomainThumbDNA polymeraseThumb domain:1. Not directly involved incatalysis2. Interacts with th

15、esynthesized DNA tomaintain correct positionof the primer and theactive site, and tomaintain a strongassociation between DNAPol and its substrate.DNA polymerase areprocessive enzymes Processivity (持续合成能力)Processivity is a characteristic ofenzymes that operate on polymericsubstrates. For DNA polymera

16、se, the degree ofprocessivity is defined as theaverage number of nucleotides addedeach time the enzyme binds aprimer:template junction(a few50,000).DNA polymerasessynthesis DNA in aprocessive mannerStructure of a sliding DNA clampSliding DNA clamps encircle the newly replicated DNAproduced by an ass

17、ociated DNA polymerase.Sliding clamps dramatically increase DNApolymerase processivity (持续合成能力)The composition of theDNA Pol III holoenzymeThree enzymes: Two copies of the DNA Pol III core enzyme One copy of the -complexThe “trombone” model for coordinatingreplication by two DNA polymerase atthe E.

18、coli replication forkSteps in the synthesiof the lagging strandRemoval of RNA primers from newlysynthesized DNARNAse H removes all of the RNAprimer except the ribonucleotidedirectly linked to the DNA end.An exonuclease removes the finalribonucleotide.DNA polymerase fills the gap, leaving aa break in

19、 the backbone between the3OH and 5 phosphate of the repairedstrand.DNA ligase repaires this “nick”.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。ter3. 复制的终止原核生物DNA的复制特点OriginTerminusDaughterDNA structure细菌染色体的复制是作为一个单位从唯一的复制起点开始，双向进行的。DNA聚合酶大肠杆菌中主要有DNA聚

20、合酶I、II、III 、IV和V。Arthur KornbergDiscovered an enzyme in E. colithat could catalyse the synthesisof DNA He called it DNA Polymerase Now known as DNA polymerase I 1959 - Nobel prize in Physiologyor Medicine的主要聚合酶，它有 3 5核酸外切酶活性,保证了DNA复制的准确性。DNA Polymerase IDNA聚合酶I (coded by polA)不是复制大肠杆菌染色体它的5 3核酸外切酶活性

21、也可用来除去冈崎片段5端RNA引物，使冈崎片段间缺口消失，保证连接酶将片段连接起来。低，只有DNA聚合酶I的5%，所以也不是复制中主要的酶。生理功能主要是起修复DNA的作用。DNA Polymerase IIDNA聚合酶II (coded by polB)的活性很 DNA聚合酶III (coded by polC)包含有7种不同的亚单位和9个亚基，其生物活性形式为二聚体。Core enzyme & holoenzymeDNA Polymerase III 它的聚合活性较强，为DNA聚合酶I的15倍，聚合酶II的300倍。 它能在引物的3OH上以每分钟约5万个核苷酸的速率延长新生的DNA

22、链，是大肠杆菌DNA复制中链延长反应的主导聚合酶。DNA Polymerase IIIThe holoenzyme consists of:The core enzyme ,The sliding clamp - The clamp loader complex A processivity switch - DNA聚合酶I(DNA损伤的修复）DNA聚合酶I、II、III(a proofreading activity)DNA聚合酶III DNA聚合酶IV和V分别由dinB和umuD2C基因编码， 主要在SOS修复过程中发挥功能。DNA Polymerase IV& V1、都以dNTP

23、为底物。2、都需要Mg2+激活。3、聚合时必须有模板链和具有3-OH末端的引物链。4、链的延伸都方向为53。DNA聚合酶的共同点：真核生物DNA的复制真核生物DNA的复制特点 有多处复制起始点； 在全部完成复制之前，各个起始点上DNA的复制不能再开始； DNA复制只在S期进行。 复制子相对较小，为40-100千碱基对。真核生物DNA的复制子被称为ARS(autonomously replicating sequences)，长约150bp左右，包括数个复制起始必需的保守区。真核生物DNA复制的起始需要起始点识别复合物（originrecognition complex，ORC）参与，ORC结合

24、于ARS，它是由6种蛋白质组成的启动复合物。真核生物DNA复制叉的移动速度大约只有50bp/秒。因此，人类DNA中每隔30,000-300,000bp就有一个复制起始位点。 已发现的真核生物DNA聚合酶有15种以上。 在哺乳动物细胞中主要有 5 种DNA聚合酶，分别称为DNA聚合酶、和 。性质DNA聚合酶DNA聚合酶DNA聚合酶DNA聚合酶DNA聚合酶亚基数4122-31在细胞内分布核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(?)3 5 外切无无有有有5 3 外切无无无无无真核生物DNA聚合酶的特性比较DNA聚合酶主要参与引物合成。DNA聚

25、合酶活性水平稳定，主要在DNA损伤的修复中起作用。DNA聚合酶主要负责DNA的复制。DNA聚合酶与后随链合成有关，在DNA合成过程中核苷切除以及碱基的切除修复中起着重要的作用。DNA聚合酶在线粒体DNA的复制中发挥作用。真核生物中还存在、和等几种DNA聚合酶，它们承担着修复损伤的功能，但这些修复酶的忠实性都很低。Key Concepts A replication fork has 1 complex of DNApolymerase /primase and 2 complexes ofDNA polymerase and/or . The DNA polymerase /primase c

26、omplexinitiates the synthesis of both DNA strands. DNA polymerase elongates the leadingstrand and a second DNA polymerase orDNA polymerase elongates the laggingstrand.复制的几种主要方式环状DNA双链的复制(1)型(2) 滚环型(rolling circle)(3) D-环型(D-loop)大肠杆菌质粒DNA双向复制的模式与实验验证。左：形复制模式图；右：3H标记质粒DNA合成图。环状DNA双链的复制- 型环状DNA可以通过滚环式

27、复制产生多单元单链DNA双链环状DNA复制起始于某一条DNA链上新生DNA链延伸，不断取代母链复制一圈，产生单位长度的线性DNA链若复制继续进行，可产生多单元线性DNA链单向复制的特殊方式X174的双链DNA复制型(RF)滚环式复制首先在动物线粒体中被发现。一条短RNA与一条DNA互补，取代了该区域原来的互补的DNA链，使得两条链的合成高度不对称，一条链上迅速合成出互补链，另一条链则为游离的单环。D loops单向复制的特殊方式DNA修复(DNA Repair)Mutagenesis(诱变)ReplicationFidelity(复制的精确性)Mutagens(诱变剂)MutationMuta

28、tion（突变）Mutation: Permanent, heritable alterationsin the base sequence of DNA.The substitution mutation(替换突变)Transition(转换): Purineor pyrimidine is replacedby the other.Transversion(颠换): apurine is replaced by apyrimidine or vice verse.无义突变错义突变沉默突变Deletion or Insertion（缺失或插入）The elimination or addit

29、ion of one ormore nucleotides in a DNA region.It may cause frameshift (移码突变)producing a non-functional protein.Real examples of deletion mutationswhich cause diseasesIn CFTR geneIn CFTR gene囊性纤维跨膜电导调节因子In FIX gene凝血因子IX9号In APC gene结肠腺癌性息肉Serious Consequences of Insertion MutationReplication fidelit

30、y(复制的精确性)Mutation relevantSpontaneous errors in DNA replication is very rare,one error per 1010 base in E. coli, which is muchless than the error that DNA polymerase IIIintroduced.Important for preserve the geneticinformation from one generation to thenextThis increased accuracy in vivo is largely d

31、ue to theproofreading function of E. coli DNA polymerasesProofreading refers to any mechanismfor correcting errors in protein ornucleic acid synthesis that involvesscrutiny of individual units after theyhave been added to the chain.Proofreading function of E. coli DNA polymerasesExperimental demonst

32、ration of the proofreadingfunction of E. coli DNA polymerase I.See A. Kornberg and T. A. Baker, 1992, DNA Replication, 2d ed., W. H. Freeman and Company.Proofreading function of E. coli DNA polymerasesWhen an incorrect base is incorporated during DNAsynthesis, the polymerase pauses, then transfers t

33、he 3 endof the growing chain to the exonuclease site where themispaired base is removed. Then the 3 end flips back intothe polymerase site, where this region is copied correctly.Proofreading play a critical role in maintainingsequence fidelity during replication E. coli DNA polymerase I (3 5exonucle

34、ase activity ) and DNA polymeraseIII ( subunit of the core polymerase )have proofreading activity. In animal cells, both of the and DNApolymerases, but not the polymerase,have proofreading activity.由于染色体DNA在生命过程中占有至高无上的地位，DNA复制的准确性以及DNA日常保养中的损伤修复就有着特别重要的意义。DNA的修复(DNA Repair)DNA修复系统功 能错配修复(mismatch r

35、epair)修复错配切除修复(碱基、核苷酸切除修复)（Base-excision repair）(nucleotide-excision repair)切除突变的碱基和核苷酸片段重组修复(recombinant repair)复制后的修复，重新启动停滞的复制叉DNA直接修复(direct repair)修复嘧啶二体或甲基化DNASOS系统(细胞DNA受到损伤或复制系统受到抑制时，细胞为求生存而产生的一种应急措施)诱导DNA损伤修复、诱变效应、细胞分裂的抑制以及溶原性细菌释放噬菌体等，导致变异大肠杆菌中DNA的修复系统DNA的转座(DNA Transposition)DNA的转座，或称移位(tr

36、ansposition)，是由可移位因子（transposable element）介导的遗传物质重排现象。A transposon (transposable element)is a DNA sequence able to insert itself (ora copy of itself) at a new location in thegenome, without having any sequencerelationship with the target locus.转座子的发现1951年，通过对玉米籽粒色斑不稳定遗传现象，B.McClintock(美)首次提出转座子的概念。D

37、s-Ac系统(Dissociation-Activation System )： 抑制因子（inhibitor, I)，后改称为解离因子(dissociator, Ds)。 激活因子(activator, Ac), 能够控制Ds因子。转座子的分类和结构特征转座子（transposon，Tn）是存在于染色体DNA上可自主复制和位移的基本单位。包括以下三类：1.插入序列（insertional sequence，IS）2. 复合式转座子（composite transposon）3. TnA家族插入序列（insertional sequence, IS）1. 最简单的转座子,不含有任何宿主基因。2. 是很小的DNA片段(约1kb)，末端具有倒 置重复序列。3. 转座时常复制宿主靶位点415bp的DNA形成正向重复区。4. 大部分IS序列只有一个开放读码框。5. 是细菌染色体或质粒DNA的正常组成部分。1.是一类带有某些抗药性基因(或其他宿主基因)的转座子复合式转座子（composite transposon）2. 两翼往往是两个相同