分子生物学chapter8

1、Chapter 8 DNA ReplicationChapter 8 DNA 复制复制DNA ReplicationGenetic 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 ar

2、e essentially perfect replicas of the parent cell. 遗传信息也遗传信息也从一个世代从一个世代到另一个世代到另一个世代进行传递。进行传递。生命至关重要的特性是：生命至关重要的特性是：即使是最复杂的生命也即使是最复杂的生命也能够被复制出来。例如，能够被复制出来。例如，当你的细胞进行有丝分当你的细胞进行有丝分裂时，它能产生两个本裂时，它能产生两个本质上是母细胞完美复制质上是母细胞完美复制品的新细胞。品的新细胞。basic conceptDNA replication:亲代亲代dsDNA在在DNA聚合酶的作用聚合酶的作用下，分别以下，分别以ssDNA为

3、模板，为模板，聚合聚合与自身碱基互补配与自身碱基互补配对的游离的对的游离的dNTP，合成出合成出两条与亲代两条与亲代DNA完全相完全相同的子代同的子代DNA分子的过程。分子的过程。Replicon（复制子）复制子） A unit of the genome in which DNA contain a region from origin to terminatorReplisome(复制体复制体) The multiprotein(about30) structure that assembles at replicating fork to undertake synthesis of D

4、NA.8.1 General features of DNA replication8.1 Semi-ConservativeReplication / 半保留复制半保留复制Semi-conservative replication: A style of DNA replication in which produces a DNA with one strand from the parent, and one newly synthesized strand.半保留复制：半保留复制：一种一种DNA的的复制方式，产生的复制方式，产生的DNA中一中一条链来自于母本、另一条链条链来自于母本、另

5、一条链是新合成的。是新合成的。8.2 Initiation of Replication / 复制的起始复制的起始OriCtwo critical repeated motifs:initial site: 13-mer motif, repeated three times, ssDNA formation during initiation.binding site: 9-mer motif for DnaA, repeated four times at oriC.8.2 Initiation of Replication / 复制的起始复制的起始E. coli replication

6、begins at a site called the OriCReplication direction New DNA enlongation from 3 5direction磷酸基团间的磷酸基团间的强负强负电性使电性使dNTP难以难以聚合聚合，5端碱基配端碱基配对困难对困难.Error base pairing.repair Bidirectional Replication / 双向复制双向复制DNA的双向复制（的双向复制（Replication is bidirectional）The first issue concerns the origin of replication.

7、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 chromosome?Second ,once replication begins, does it proceed in a single direction or

8、 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 the helix are unwound, creating a replication fork. Such a fork initially appears at th

9、e 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. Cairns： Replication model（1963）8.3 Semi-discontinuous replicationSemi-discontinuous repl

10、ication / 半不连续复制半不连续复制The continuous strand, or leading strand, is the one in which 53synthesis proceeds in the same direction as replication fork movement.The discontinuous strand or lagging strand, is the one in which 53synthesis proceeds in the direction opposite to the direction of fork movement

11、. and, is the oneLeading strand& lagging strandLeading strand& lagging strand8.4 Elongation of Replicationand its Proteins复制延伸及其相关蛋白复制延伸及其相关蛋白8.4.1 Helicase and SSBs8.4.2 DNA polymerase III8.4.3 Explanation for 35 Synthesis8.4.4 Primers8.4.1 解旋酶与解旋酶与SSB8.4.2 DNA聚合酶聚合酶III8.4.3 关于关于35合成合成8.4.4

12、 引物引物8.4.1 Helicase and SSBs / 解旋酶与解旋酶与SSBDNA Helicases解解链酶链酶：Unwind the DoubleHelix in Advance of the Replication ForkHelicase: the enzyme harness the chemical energy of ATP to separate parental DNA strands at replication fork.The helicase is encoded by the dnaB gene.Moves along ssDNA in a defined

13、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 StabilizeSingled-stranded DNA Prior to ReplicationSingle stranded Binding Proteins1.binding much more strongly to single-strand tha

14、n to double strand DNA.2.aid helicase action by binding tightly andcooperatively to newly formed single-stranded DNA and keeping it from annealing with its partner.3.by coating the single-stranded DNA, SSBs also protect it from degradation.4.Binding of one SSB promotes the binding of another SSB to

15、the adjacent ssDNA, This is called cooperative binding.SSB: single-strand DNA binding proteinssDNAssDNAFile: E coli SSB bound to ssDNA 1EYG.val8.4.2 DNA polymerase / DNA聚合酶聚合酶 DNA polymerase I DNA polymerase II DNA polymerase IIIE. coli DNA polymerase IPurification and characterization by Arthur Kor

16、nberg in1955a single-polypeptide enzyme, 928 amino acidresidues.Mr 103,000; encoded by the polA gene5 3 polymerase activity: nucleotides polymerization ,1000nt/min。3-5 exonuclease activity :remove incorrect (mismatched) bases.5 -3 exonuclease activity: dsDNA(切除切除嘧啶嘧啶二聚体二聚体) remove distorted (mispair

17、ed) segments; excision the primer. Polymerase I : three Active Sites on Its Single Polypeptide Chain Hans Klenow 用枯草杆菌蛋白用枯草杆菌蛋白酶酶（subtilisin）或胰蛋白或胰蛋白酶酶（trypsin）进进行降解，行降解， DNA聚合聚合酶酶I被切成两个片段。被切成两个片段。smaller fragment: 5- 3exonuclease activitylarger fragment :Klenow fragment has the polymerase and 3- 5e

18、xonuclease activities.DNA Polymerase 多亚基酶，分子量：多亚基酶，分子量：120kD，每个每个Ecoli.细胞细胞100个酶分子。个酶分子。催化活性：催化活性： 5 3 聚合酶活性聚合酶活性，但活力低但活力低 ,只有只有DNA Polymerase I 的的5；3 5 外切酶活性外切酶活性。其它生理功能尚不清楚，可能在可能在DNA的修复中起某些的修复中起某些作用作用。 DNA polymerase III / DNA聚合酶聚合酶IIIDNA polymerase III is used for almost all of replicationThe ful

19、l collection of subunits is called the holoenzyme. The minimal collection of subunits needed for polymerization is called the DNA polymerase III coreDNA聚合酶聚合酶III 二聚体二聚体 dimerdimerCatalytic core: Subunit-has the DNA polymerase activity. Subunit-3-5 proofreading exonuclease activitySubunit-stimulate e

20、xonuclease Its two subunits clamp around the DNA template and also bind to the core, preventing it from falling off the template. Sliding clamp / 滑行夹滑行夹donut shaped protein little processivity10 bp before detaching from the templateSliding clamp / 滑行夹滑行夹The core polymerase alone is able to polymeriz

21、e DNA, but it has very 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, preventi

22、ng it from falling off 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 / 攀岩安全扣攀岩安全扣The function of sliding clamp is just like the safety clamp being used in activit

23、ies like climbing.structure of sliding clampFile: E coli DNA pol III beta dimer 1MMI.valClamp loader / 滑行夹加载器滑行夹加载器Iit is necessary to attach the sliding clamp onto the DNA. structure of clamp loaderFile: E coli clamp loader gamma complex of DNA pol III.valDNA连接酶连接酶ligase所需能量所需能量:NAD (大肠杆菌等细菌大肠杆菌等细菌

24、) ATP（动物细胞和噬菌体）（动物细胞和噬菌体）催化催化dsDNA切口处的切口处的5-磷酸和磷酸和3-羟基生成羟基生成磷酸二酯键；磷酸二酯键；Proofreading / 校正校正3-5 exonuclease activity3-5外切核酸酶活性外切核酸酶活性亚基和亚基Proofreading / 校正校正8.4.3 Explanation for 35 Synthesis关于关于35合成合成53 Synthesis continues after proofreading35 Synthesis stops after proofreading 53 Synthesis continue

25、s after proofreading53 Synthesis continues after proofreading35 Synthesis 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

26、can not perform the same trick with initiation of DNA polymerase.8.4.4 Primers / 引物引物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. The

27、se pieces are called primers. primers can form short RNA-DNA hybrids. The primers contain a 3 end. Primer for Okazaki fragmentIn leading strand synthesis, a primer is only needed. in lagging strand synthesis, each Okazakifragment must begin with a new primer. Thus primase must add a primer for every

28、 1-2kb of lagging strand synthesis.Short RNA primerPrimer Removal / 引物去除引物去除RNA primers 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 stre

29、tch of DNA in its place.8.5 DNA topology / DNA拓扑学拓扑学As replication proceeds, the double-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

30、 into a shape called a supercoil. 在共价闭环双螺旋基础上进一步扭转盘曲在共价闭环双螺旋基础上进一步扭转盘曲, ,形形成成麻花状的超螺旋麻花状的超螺旋(supercoil)(supercoil)，体积进一步体积进一步压缩，密度较大压缩，密度较大。Tertiary structure -superhelix DNA 在一端使绳子向在一端使绳子向缠紧缠紧的方向的方向旋转，再将绳子两端连接起来，旋转，再将绳子两端连接起来，Leads to Left handed superhelix DNA Positive supercoiled形成过程：形成过程：B DNA le

31、ft unwinding(左旋左旋)Leads to Negative Supercoiled http:/ of forming superhelix DNA Vinogard. J(1968) Vinograd equation L - Linking number (双螺旋双螺旋DNA的的交叉数交叉数) T -Twisting number(双链双链DNA的的缠绕数缠绕数，初级双螺旋的，初级双螺旋的圈数圈数 ) W -Writhing number(扭曲数，即扭曲数，即超螺旋数超螺旋数 )L=T+W多数情况细胞内的多数情况细胞内的DNA处于处于负负超螺旋超螺旋状态。如细菌质粒、状态。如细

32、菌质粒、噬菌体、大肠杆菌染色体噬菌体、大肠杆菌染色体DNA、真核细胞染色体真核细胞染色体DNA意义意义 负负超螺旋会部分地转变为超螺旋会部分地转变为 单链泡状单链泡状结构，结构， 易于释放其易于释放其所所包含的信息包含的信息，蛋白质会与这，蛋白质会与这些单链泡状结构结合参与复制些单链泡状结构结合参与复制和转录。和转录。DNA在水溶液中，构型偏在水溶液中，构型偏B型状态型状态DNA以以10.5bp/helix 为最稳定状态为最稳定状态小于小于10.5bp/helix向向Positive supercoil发展（发展（紧缩态紧缩态）大于大于10.5bp /helix向向Negative super

33、coil发展（发展（松弛态松弛态）vIn vivo(细胞内细胞内)富含富含AT易于解易于解链，链，IR区区形成十字型，产生形成十字型，产生Negative supercoil ，以消除解链产生的应力，维持，以消除解链产生的应力，维持DNA分子的分子的稳定状态稳定状态vB-DNA Z-DNA B-DNA 调控基因表达调控基因表达DNA复制复制DNA 转录转录需需D.S S.S 状态状态 DNA分子需以高度致密的分子需以高度致密的超螺旋状态超螺旋状态压缩在核小体内压缩在核小体内Topoisomerase I / 拓扑异构酶拓扑异构酶I拓扑异构酶（拓扑异构酶（topoisomerase ）：）：参与

34、构型改变参与构型改变催化一个催化一个DNA单链或双螺旋链穿过另一单（双）链单链或双螺旋链穿过另一单（双）链. Top:催化催化DNA链断裂和重新连接，每次只链断裂和重新连接，每次只作用于作用于一条链一条链，即催化瞬时的单链断裂与连，即催化瞬时的单链断裂与连接，不需要接，不需要ATP or NAD. Remove negative superhelix. Top : 同时断裂并连接同时断裂并连接双链双链DNA，常需要常需要能量辅因子能量辅因子ATP. Induce negative superhelix. Breakage & rejoining ssDNA at phospho-die

35、ster bond Remove negative helix relax B-DNA double helix （L+1/次次）能量：能量：贮藏水解磷酸二酯键的能量贮藏水解磷酸二酯键的能量topoisomeraseDNA拓扑异构酶拓扑异构酶 ( topoisomerase -gyrase） Topoisomerases Remove Supercoils Produced by DNA Unwinding at the Replication Fork .引入引入负超螺旋负超螺旋，消除复制叉前进时带来的扭曲张力，消除复制叉前进时带来的扭曲张力四个亚基（四个亚基（A2B2）：）： B 亚基：具

36、有亚基：具有ATP酶活性酶活性A亚基：亚基： 切开、连接活性（内切酶和连接酶活）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 intact and unsynthesized between both

37、 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 and releases the other chromosome by passing it through the gap. 8

38、.6 DNA 复制复制DNA replicationInitiation of DNA replication辨认并结合起始位点辨认并结合起始位点打开双螺旋打开双螺旋防止复螺旋防止复螺旋单链结合蛋白（单链结合蛋白（SSB）解链酶解链酶引物引物引物酶引物酶DnaA合成合成Dna BDna C3 5 3 5 3 HO5引物引物酶酶 Dna B、 Dna CDNA拓扑异构酶拓扑异构酶SSBDna AInitiation of DNA replicationtwo critical repeated motifs:initial site: 13-mer motif, repeated three t

39、imes, ssDNA formation during initiation.binding site: 9-mer motif for DnaA, repeated four times at oriC.Preprimosome（前引发体）前引发体） 引发前体引发前体和和引发酶引发酶组装形成组装形成引发体引发体（primosome）才能发挥作用。）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

40、 oriC.DnaA protein 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

41、In the presence 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

42、unwinds the DNA 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 allowin

43、g one strand to rotate around the other.Eukaryotic DNA polymerases work inefficientProkaryotic DNAEukaryotic DNA1000 nt / sec1000 nt / min8.6 DNA Replication in Eukaryotes Genomes are much larger than prokaryotic genomes.Replication forks move much more slowly. this is possibly due to the histones a

44、ssociated with eukaryotic DNA Replication initiates at hundreds or thousands of different originsMultiple initiation sites多重起始位点多重起始位点electron micrograph many replication bubbles along the chromosomal DNA. eukaryotic DNA polymerasesPol (Synthesis of lagging strand)Pol (Synthesis of leading strand)Po

45、l (Function unknown)Pol (DNA repair)Pol (Replication of mitochondrial DNA)Pol IIIPol I3 prokaryotic DNA polymerasesPol II? The most important for replication are DNA polymerase and DNA polymerase . 8.6.1 Replication initiation in Eukaryotes真核生物复制起始真核生物复制起始S. cerevisiae（面包酵母）面包酵母）www2.biomed.cas.cz/b

46、enada/lem117/eng/stereo.htm ARS: autonomouslyreplicating sequence自主复制序列自主复制序列 OriCARS is important for initiation.Eukaryotic replication origins真核生物复制起点真核生物复制起点In higher eukaryotes, it has been much more difficult to pinpoint sequences that serve as origins of replication. In some cases the site of

47、the origin may vary over 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，在更高等的真核生物中，要找出作为复制起点序列要找出作为复制起点序列的确切位置要困难得多。的确切位置要困难得多。在

48、有些情况下，复制起点在有些情况下，复制起点的序列可能会相差几千个的序列可能会相差几千个碱基，在另一些情况下则碱基，在另一些情况下则看起来根本没有统一的复看起来根本没有统一的复制起点位置。这时，在决制起点位置。这时，在决定复制起点中起重要作用定复制起点中起重要作用的是一些其它因素如核小的是一些其它因素如核小体密度等，而不是碱基序体密度等，而不是碱基序列。列。The cell cycle / 细胞周期细胞周期the protein machinery that acts to initiate replication appears to be well conserved. the cell c

49、ycle 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 important that DNA be replicated only once, and only during S-phase, to avoid chaos in the genome. pre-RC: pre-Replicative Complex前复制复合体前复制复合体pre-RC The co

50、llection of proteins that initiates replication in S-phase is called the pre-replicative complex (pre-RC). origin recognition complex (ORC)bind to DNAORC attracts 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 inac

51、tive until S-phase, a protein called Cdc45 binds. Cdc45 allows the pre-RC to initiate replication by activating Mcm2-7 to function as a helicase, and recruiting DNA polymerases to the pre-RC. Inactive Cdk2Active Cdk2Cdc45Cdc45How does the Cdc45 know to only activate the pre-RC during 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 / 端粒端粒Another challenge faced by eukaryotes during synthesis is what to do