分子生物学chapter-8.ppt

1、Chapter 8 DNA Replication Chapter 8 DNA 复制,DNA Replication,Genetic information is also passed from generation to generation. A crucial property of life is that even the most complex organisms are able to be copied. When one of your cells divides by mitosis, for example, it makes two new cells that a

2、re essentially perfect replicas of the parent cell.,遗传信息也从一个世代到另一个世代进行传递。生命至关重要的特性是：即使是最复杂的生命也能够被复制出来。例如，当你的细胞进行有丝分裂时，它能产生两个本质上是母细胞完美复制品的新细胞。,basic concept,DNA replication:亲代dsDNA在DNA聚合酶的作用下，分别以ssDNA为模板，聚合与自身碱基互补配对的游离的dNTP，合成出两条与亲代DNA完全相同的子代DNA分子的过程。,Replicon（复制子）A unit of the genome in which DNA co

3、ntain a region from origin to terminator Replisome(复制体) The multiprotein(about30) structure that assembles at replicating fork to undertake synthesis of DNA.,8.1 General features of DNA replication,8.1 Semi-Conservative Replication / 半保留复制,Semi-conservative replication: A style of DNA replication in

4、 which produces a DNA with one strand from the parent, and one newly synthesized strand.半保留复制：一种DNA的复制方式，产生的DNA中一条链来自于母本、另一条链是新合成的。,8.2 Initiation of Replication / 复制的起始,OriC,two critical repeated motifs: initial site: 13-mer motif, repeated three times, ssDNA formation during initiation. binding si

5、te: 9-mer motif for DnaA, repeated four times at oriC.,8.2 Initiation of Replication / 复制的起始,E. coli replication begins at a site called the OriC,Replication direction,New DNA enlongation from 3 5direction,磷酸基团间的强负电性使dNTP难以聚合，5端碱基配对困难.,Error base pairing.repair,Bidirectional Replication / 双向复制,DNA的双

6、向复制（Replication is bidirectional）,The first issue concerns the origin of replication.Where along the chromosome is DNA replication initiated? Is there only a single origin, or does DNA synthesis begin at more than one point? Is a point of origin random or is it located at a specific region along the

7、 chromosome? Second ,once replication begins, does it proceed in a single direction or in both directions away from the origin? In other words, is replication unidirectional or bidirectional?,Replication fork : at the actual point along the chromosome where replication is occurring, the strands of t

8、he helix are unwound, creating a replication fork.Such a fork initially appears at the point of origin of synthesis and then moves along the DNA duplex as replication proceeds. If replication is bidirectional, two such forks will be present, migrating in opposite directions away from the origin.,Cai

9、rns： Replication model（1963）,8.3 Semi-discontinuous replication,Semi-discontinuous replication / 半不连续复制,The continuous strand, or leading strand, is theone in which 53synthesis proceeds in the samedirection as replication fork movement. The discontinuous strand or lagging strand, isthe one in which

10、53synthesis proceeds in the direction opposite to the direction of fork movement. and, is the one,Leading strand& lagging strand,Leading strand& lagging strand,8.4 Elongation of Replication and its Proteins 复制延伸及其相关蛋白,8.4.1 Helicase and SSBs 8.4.2 DNA polymerase III 8.4.3 Explanation for35 Synthesis

11、 8.4.4 Primers,8.4.1 解旋酶与SSB 8.4.2 DNA聚合酶III 8.4.3 关于35合成8.4.4 引物,8.4.1 Helicase and SSBs / 解旋酶与SSB,DNA Helicases解链酶：Unwind the Double Helix in Advance of the Replication Fork,Helicase: the enzyme harness the chemical energy of ATP to separate parental DNA strands at replication fork. The helicase i

12、s encoded by the dnaB gene. Moves along ssDNA in a defined direction. DNA helicases can have a polarity of either 53 or 3 5 Helicase is the key protein of DNA replication, recombination, repair.,Single stranded Binding Proteins Stabilize Singled-stranded DNA Prior to Replication,Single stranded Bind

13、ing Proteins,1.binding much more strongly to single-strand than to double strand DNA. 2.aid helicase action by binding tightly and cooperatively to newly formed single-stranded DNAand keeping it from annealing with its partner. 3.by coating the single-stranded DNA, SSBs also protect it from degradat

14、ion. 4.Binding of one SSB promotes the binding ofanother SSB to the adjacent ssDNA, This is called cooperative binding.,SSB: single-strand DNA binding protein,ssDNA,ssDNA,File: E coli SSB bound to ssDNA 1EYG.val,8.4.2 DNA polymerase / DNA聚合酶,DNA polymerase IDNA polymerase IIDNA polymerase III,E. col

15、i DNA polymerase I,Purification and characterization by Arthur Kornberg in1955 a single-polypeptide enzyme, 928 amino acid residues. Mr 103,000; encoded by the polA gene,5 3 polymerase activity: nucleotides polymerization ,1000nt/min。 3-5 exonuclease activity :remove incorrect (mismatched) bases. 5

16、-3 exonuclease activity: dsDNA(切除嘧啶二聚体) remove distorted (mispaired) segments; excision the primer.,Polymerase I : three Active Sites on Its Single Polypeptide Chain,Hans Klenow 用枯草杆菌蛋白酶（subtilisin）或胰蛋白酶（trypsin）进行降解， DNA聚合酶I被切成两个片段。 smaller fragment: 5- 3exonuclease activity larger fragment :Klenow

17、 fragment has the polymerase and 3- 5exonuclease activities.,DNA Polymerase 多亚基酶，分子量：120kD，每个Ecoli.细胞 100个酶分子。 催化活性：5 3 聚合酶活性，但活力低 ,只有DNA Polymerase I 的5； 3 5 外切酶活性。 其它生理功能尚不清楚，可能在DNA的修复中起某些作用。,DNA polymerase III / DNA聚合酶III,DNA polymerase III is used for almost all of replication,The full collectio

18、n of subunits iscalled the holoenzyme.,The minimal collection of subunits needed for polymerization is called the DNA polymerase III core,DNA聚合酶III 二聚体 dimer,Catalytic core: Subunit-has the DNA polymerase activity. Subunit-3-5 proofreading exonuclease activity Subunit-stimulate exonuclease,Its two s

19、ubunits clamp around the DNA template and also bind to the core, preventing it from falling off the template.,Sliding clamp / 滑行夹,donut shaped protein,little processivity,10 bp before detaching from the template,Sliding clamp / 滑行夹,The core polymerase alone is able to polymerize DNA, but it has very

20、 little processivity.,The core polymerase can only polymerize approximately 10 nucleotides before detaching from the template.,The subunit(sliding clamp), is a donut shaped protein,like helicase. Its two subunits clamp around the DNA template and also bind to the core, preventing it from falling off

21、 the template., Dimer is ring shaped, assembly or removed required energy. Dimer bound to DNA but slides along Dimer endows holoenzyme with highly processivity.,Safety clamp for climbing / 攀岩安全扣,The function of sliding clamp is just like the safety clamp being used in activities like climbing.,struc

22、ture of sliding clamp,File: E coli DNA pol III beta dimer 1MMI.val,Clamp loader / 滑行夹加载器,Iit is necessary to attach the sliding clamp onto the DNA.,structure of clamp loader,File: E coli clamp loader gamma complex of DNA pol III.val,DNA连接酶ligase,所需能量: NAD(大肠杆菌等细菌) ATP （动物细胞和噬菌体）,催化dsDNA切口处的5-磷酸和3-羟基

23、生成磷酸二酯键；,Proofreading / 校正,3-5 exonuclease activity 3-5外切核酸酶活性,亚基和亚基,Proofreading / 校正,8.4.3 Explanation for 35 Synthesis 关于35合成,53 Synthesis continues after proofreading35 Synthesis stops after proofreading,53 Synthesis continues after proofreading,53 Synthesis continues after proofreading,35 Synth

24、esis stops after proofreading,35 Synthesis stops after proofreading,RNA polymerase initiates transcription simply by starting a new RNA chain; it puts the first nucleotide in place and then joins the next to it. But DNA polymerase can not perform the same trick with initiation of DNA polymerase.,8.4

25、.4 Primers / 引物,We now know that the missing component here is a primer. The primer is not DNA, but a short piece of RNA.,What is missing?,primase, puts down short about 10 nucleotides long pieces of RNA at sites of replication. These pieces are called primers. primers can form short RNA-DNA hybrids

26、. The primers contain a 3 end.,Primer for Okazaki fragment,In leading strand synthesis, a primeris only needed.,in lagging strand synthesis, each Okazaki fragment must begin with a new primer.,Thus primase must add a primer for every 1-2kb of lagging strand synthesis.,Primer Removal / 引物去除,RNA prime

27、rs must be replaced with DNA before replication is completed.,DNA polymerase I has 53 exonuclease activity and degrade primer beginning at the 5end. Simultaneously,it replaces the primer by synthesizing a short stretch of DNA in its place.,8.5 DNA topology / DNA拓扑学,As replication proceeds, the doubl

28、e-stranded DNA is increasingly structure of DNA and the circular shape of the E. coli chromosome,this creates a high degree of tension ahead of the replication fork. The tension relieves itself by tangling the DNA into a shape called a supercoil.,在共价闭环双螺旋基础上进一步扭转盘曲,形成麻花状的超螺旋(supercoil)，体积进一步压缩，密度较大。

29、,Tertiary structure -superhelix DNA,在一端使绳子向缠紧的方向旋转，再将绳子两端连接起来，Leads to Left handed superhelix DNA Positive supercoiled,形成过程：,B DNA left unwinding(左旋) Leads to Negative Supercoiled,http:/ of forming superhelix DNA,Vinogard. J(1968) Vinograd equation L - Linking number (双螺旋DNA的交叉数) T -Twisting number(

30、双链DNA的缠绕数，初级双螺旋的圈数 ) W -Writhing number(扭曲数，即超螺旋数 ),L=T+W,多数情况细胞内的DNA处于负超螺旋状态。如细菌质粒、噬菌体、大肠杆菌染色体DNA、真核细胞染色体DNA 意义负超螺旋会部分地转变为 单链泡状结构， 易于释放其所包含的信息，蛋白质会与这些单链泡状结构结合参与复制和转录。,DNA在水溶液中，构型偏B型状态 DNA以10.5bp/helix 为最稳定状态 小于10.5bp/helix向Positive supercoil发展（紧缩态） 大于10.5bp /helix向Negative supercoil发展（松弛态）,In vivo(

31、细胞内) 富含AT易于解链，IR区形成十字型，产生Negative supercoil ，以消除解链产生的应力，维持DNA分子的稳定状态 B-DNA Z-DNA B-DNA 调控基因表达 DNA复制 DNA 转录,需D.S S.S 状态,DNA分子需以高度致密的超螺旋状态压缩在核小体内,Topoisomerase I / 拓扑异构酶I,拓扑异构酶（topoisomerase ）：参与构型改变催化一个DNA单链或双螺旋链穿过另一单（双）链.,Top:催化DNA链断裂和重新连接，每次只作用于一条链，即催化瞬时的单链断裂与连接，不需要ATP or NAD. Remove negative super

32、helix. Top : 同时断裂并连接双链DNA，常需要能量辅因子ATP. Induce negative superhelix.,Breakage & rejoining ssDNA at phospho-diester bond Remove negative helix relax B-DNA double helix （L+1/次）,能量：贮藏水解磷酸二酯键的能量,topoisomerase,DNA拓扑异构酶 ( topoisomerase -gyrase）,Topoisomerases Remove Supercoils Produced by DNA Unwinding at t

33、he Replication Fork .,引入负超螺旋，消除复制叉前进时带来的扭曲张力,四个亚基（A2B2）： B 亚基：具有ATP酶活性 A亚基： 切开、连接活性（内切酶和连接酶活）,Type II topoisomerases are also important for the termination of replication.,the replication forks proceed until they reach a set of termination sequences .Only a small region of the original DNA remains i

34、ntact and unsynthesized between both forks.,Then this region is denatured, each single strand can be used as a template to fully complete replication.,The two daughter chromosomes are interconnected.topoisomerase II makes a double stranded break in one of the chromosomes andreleases the other chromo

35、some by passing it through the gap.,8.6 DNA 复制 DNA replication,Initiation of DNA replication,辨认并结合起始位点,打开双螺旋,防止复螺旋,单链结合蛋白（SSB）,解链酶,引物,引物酶,DnaA,合成,Dna B,Dna C,3,5,3,5,引物酶,Dna B、 Dna C,DNA拓扑异构酶,SSB,Dna A,Initiation of DNA replication,two critical repeated motifs: initial site: 13-mer motif, repeated t

36、hree times, ssDNA formation during initiation. binding site: 9-mer motif for DnaA, repeated four times at oriC.,Preprimosome（前引发体）,引发前体和引发酶组装形成引发体（primosome）才能发挥作用。,DnaA protein is initiation factor，which recognized and bind to the four repeated 9-mer sequence on right side of the oriC. DnaA protein

37、 binding is cooperative； once the four 9-bp repeats are occupied,20-40 additional DnaA monomers bind the entire oriC region. HU: general DNA binding protein. Similar to histones. Causes DNA to bend and fold into structure resembling beaded chromatin.,Creating replication fork at oriC,In the presence

38、 of ATP, the DnaA protein mediates the separation of the strands of the DNA duplex by acting on three AT-rich 13-mer tandem repeats and form an open complex. Each DnaB-DnaC complex consists of 6 DnaC monomers bound to a hexamer of DnaB. DnaB protein has helicase activity and it further unwinds the D

39、NA in both directions. The protein SSB stabilizes the single stranded DNA as it is formed.,DnaB activates a DnaG primase, in one case to initiate the leading strand, and in the other to initiate the first Okazaki fragment of the lagging strand. Gyrase 促旋酶: acts as a swivel enzyme allowing one strand

40、 to rotate around the other.,Eukaryotic DNA polymerases work inefficient,Prokaryotic DNA,Eukaryotic DNA,1000 nt / sec,1000 nt / min,8.6 DNA Replication in Eukaryotes,Genomes are much larger than prokaryotic genomes.,Replication forks move much more slowly. this is possiblydue to the histones associa

41、ted with eukaryotic DNA,Replication initiates at hundreds or thousands of different origins,Multiple initiation sites 多重起始位点,electron micrograph,many replication bubbles along the chromosomal DNA.,eukaryotic DNA polymerases,Pol (Synthesis of lagging strand)Pol (Synthesis of leading strand)Pol (Funct

42、ion unknown)Pol (DNA repair)Pol (Replication of mitochondrial DNA),Pol III,Pol I,3 prokaryotic DNA polymerases,Pol II,?,The most important for replication are DNApolymerase and DNA polymerase .,8.6.1 Replication initiation in Eukaryotes 真核生物复制起始,S. cerevisiae（面包酵母）,www2.biomed.cas.cz/benada/lem117/e

43、ng/stereo.htm,ARS: autonomously replicating sequence 自主复制序列,OriC,ARS is important for initiation.,Eukaryotic replication origins 真核生物复制起点,In higher eukaryotes, it has been much more difficult to pinpoint sequences that serve as origins of replication. In some cases the site of the origin may vary ov

44、er thousands of bases, in other cases there seem to be no consistent sites of replication origin. Factors other than base sequence, such as nucleosome density, may play important roles in determining the origin.,在更高等的真核生物中，要找出作为复制起点序列的确切位置要困难得多。在有些情况下，复制起点的序列可能会相差几千个碱基，在另一些情况下则看起来根本没有统一的复制起点位置。这时，在决

45、定复制起点中起重要作用的是一些其它因素如核小体密度等，而不是碱基序列。,The cell cycle / 细胞周期,the protein machinery that acts to initiate replication appears to be well conserved.,the cell cycle is often divided into four phases: G1, a growth phase; S, a DNA synthesis phase; G2, another growth phase; and M, mitosis.,It is critically i

46、mportant that DNA be replicated only once, and only during S-phase, to avoid chaos in the genome.,pre-RC: pre-Replicative Complex 前复制复合体,The collection of proteins that initiates replication in S-phaseis called the pre-replicative complex (pre-RC).,origin recognition complex (ORC)bind to DNA,ORC att

47、racts the Cdc6 and Cdt1,which in turn attract the helicase Mcm2-7.,DNA synthesis does not occur.,The complex remains attached to the DNA but inactive until S-phase, a protein called Cdc45 binds. Cdc45 allows thepre-RC to initiate replication by activating Mcm2-7 to function as a helicase, and recrui

48、ting DNA polymerases to the pre-RC.,Inactive Cdk2,Active Cdk2,Cdc45,Cdc45,How does the Cdc45 know to only activate the pre-RCduring S-phase?,During the G1 phase, Cdk2 is inactive; however it becomes active during S phase.,Active Cdk2 is necessary for Cdc45 to be able to bind to the pre-RC.,8.6.2 Telomeres / 端粒,Another challenge faced by eukaryotes during synthesis is what to do with primers at the end of the chromosome. The end of a eukaryotic chromosome is called the telomere.,真核生物DNA合成面临的另一个挑战是如何处理位于染色体末端的引物。真核生物染色体的末端称为端粒。,telomere,