第4章DNA复制

1、Section E DNA ReplicationMolecular Biology CourseE3: Eukaryotic DNA replicationMolecular Biology CourseE1: DNA Replication: An OverviewReplicons, semi-conservative, semi-discontinous, RNA primingE2: Bacterial DNA replicationExperimental system, initiation, unwinding, elongation, termination & se

2、gregationExperimental system, cell cycle, initiation, replication forks, nuclear matrix, telomere repl.DNA replicationE1: DNA Replication: An Overview 复制子复制子Replicons 半保留复制半保留复制semi-conservative 半不连续复制半不连续复制semi-discontinous RNA引导引导RNA priming复制子：以单一单位复制的任一段复制子：以单一单位复制的任一段DNA片段。含片段。含有一个复制起始点有一个复制起始点

3、Origin与终止点与终止点Terminus 。Origin is the DNA sequence where a replicon initiates its replication.Terminus is the DNA sequence where a replicon usually stops its replication DNA replicationE1-1 Replicons How many replicons?原核基因组原核基因组Prokaryotic genome: a single circular DNA = a single replicon真核基因组真核基因组

4、Eukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosomeDNA replication原核基因组原核基因组Bidirectional replication of a circular bacterial replicon All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circullar and comprise single replicons. There

5、is a single termination site roughly 180o opposite the unique origin. DNA replicationLinear viral DNA molecules usually have a single origin, replication details (see Section R)In all the cases, the origin is a complex region where the initiation of DNA replication and the control of the growth cycl

6、e of the organism are regulated and co-ordinated. (OK! Make sense. Example ?)DNA replicationThe long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own origin. A typical mammalian cell has 50000-100000replicons with a size range of 40-200 kb. Where replicat

7、ion forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required 真核基因组真核基因组Multiple eukaryotic replicons and replication bubblesDNA replicationDNA replicationMultiple eukaryotic replicons and replication bubblesreplication bubbles re

8、plication forkIs there any problem with the above replication model? DNA replicationE1-2 Replication is Semi-conservative 半保留复制半保留复制 Semi-conservative mechanism15N labeling experiment15N labeling: grow cells in ?Collect DNA: grow cells in ?Separation: method ?Result interpretation15N labeled DNAunla

9、beled DNADNA replicationDNA replicationE1-3 Replication is Semi-discontinuous 半不连续复制半不连续复制 Semi-discontinuous replicationLigation DNA replicationOkazaki fragments冈崎片段冈崎片段Discovery of Okazaki fragmentsEvidence for semi-discontinuous replication3H thymidine pulse-chase labeling experimentGrow E. coliA

10、dd 3H thymidine in the medium for a few second spin down and break the cell to stop labeling analyze found a large fraction of nascent DNA (1000-2000 nt) = Okazaki fragments1. Grow the cell in regular medium then analyze the small fragments join into high molecular weight DNA = Ligation of the Okaza

11、ki fragmentsDNA replicationDNA replicationE1-4 RNA priming RNA引导引导 The first few nucleotides at the 5-end of Okazaki fragments are ribonucleotides. Hence, DNA synthesis is primed by RNA that is then removed before fragments are joined. Crucial for high fidelity of replicationDNA replicationE1: DNA R

12、eplication: An Overview 复制子复制子Replicons 半保留复制半保留复制semi-conservative 半不连续复制半不连续复制semi-discontinous RNA引导引导RNA primingDNA replicationE2: 原核生物原核生物细菌细菌DNA的复制的复制 实验系统实验系统 Experimental system 起始起始 initiation 解旋解旋 unwinding 延伸延伸 elongation 终止与分离终止与分离termination & segregationE2-1: 体外实验系统体外实验系统In vitro e

13、xperimental systems 纯化的纯化的 DNA: E.coli 大噬菌体病毒大噬菌体病毒T4或或T7 小噬菌体病毒小噬菌体病毒 (f fX174, 5 Kb)和质粒和质粒DNA。 完成复制所需的所有的蛋白质与其它因子完成复制所需的所有的蛋白质与其它因子DNA replicationPut DNA and protein together and ask replication question 研究系统研究系统: E. coli 起始点起始点 oriC 克隆进质粒克隆进质粒 构成构成微型染色体（微型染色体（minichromosomes）方便）方便DNA复复制的研究。制的研究。D

14、NA replicationE2-2: 起始起始InitiationoriC 含四个含四个 9 bp 结合位点为起始蛋白因子结合位点为起始蛋白因子DnaA的的结合结合. DnaA的合成与生长速率相关联的合成与生长速率相关联 ，因而复制的，因而复制的起始也与生长速率相关联起始也与生长速率相关联.DnaA 形成形成 30-40分子的复合体，每分子结合分子的复合体，每分子结合1分子分子ATP, 有助于有助于3个个13bp富含富含AT序列的重复序列的解链序列的重复序列的解链形成负超螺旋，从而允许形成负超螺旋，从而允许 DnaB 结合结合. DnaB 解旋酶解旋酶：利用：利用ATP水解的能量结合并解开双

15、水解的能量结合并解开双链链DNA。 单链结合蛋白单链结合蛋白Ssb (single-stranded binding protein) ：结合到单链结合到单链DNA上上.DNA 引发酶：引发酶：结合到结合到DNA上并合成一段短的上并合成一段短的RNA引物，从而引导前导链的复制与双向复制引物，从而引导前导链的复制与双向复制。引发酶体引发酶体: DnaB 解旋酶与解旋酶与DNA引发酶引发酶Initiation细菌复制的再起始作用细菌复制的再起始作用 正超螺旋的形成：正超螺旋的形成：复制叉处的螺旋转角的去复制叉处的螺旋转角的去除会导致分子其余位额转角的导入。除会导致分子其余位额转角的导入。正超螺旋的

16、解除：正超螺旋的解除：通过通过II型拓扑异构酶即型拓扑异构酶即DNA旋转酶。旋转酶。DNA replicationE2-3: 解旋解旋UnwindingDNA replicationE2-4: 延伸延伸Elongation RNA引物的合成：引发体引导。引物的合成：引发体引导。2. DNA链的延伸：链的延伸：DNA聚合酶聚合酶III全酶全酶（ -subunit-polymerase；-subunit-35 proofreading exonuclease ；subunit-clamp the polymerase ）。 3.引物去除，填补引物去除，填补DNA：DNA聚合酶聚合酶I。4. 冈崎片

17、段的连接：冈崎片段的连接：DNA连接酶。连接酶。Elongation: lagging strand replication Polymerase III holoenzyme(DNA pol III)DNA pol I (53 exonuclease activity)DNA pol I (53 polymerase activity)DNA ligaseDNA replicationE2-5: 终止与分离终止与分离Termination and Segregation终止点终止点: containing several terminator sites (ter) approximatel

18、y 180o opposite oirC.Tus 蛋白蛋白: ter binding protein, an inhibitor of the DnaB helicase终终 止止Terminationter拓扑异构酶拓扑异构酶IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.分分 离离SegregationDNA replicationE3: 真核生物真核生物DNA的复制的复制 细胞周期细胞周期 cell cycle实验系统实验系统 Experimental system,

19、 起始起始 initiation, 复制叉复制叉 replication forks, 核基质核基质 nuclear matrix, 端粒的复制端粒的复制 telomere replication.G1G1 preparing for DNA replication (cell growth)S SDNA replicationG2G2a short gap before mitosisM Mmitosis and cell divisionEntry into the S-phase:CyclinsCyclin-dependent protein kinases (CDKs)signalin

20、gE3: Cell cycle When to replicate？E3-1: 体外体外 In vitro 实验系统实验系统 纯化的纯化的 DNA : 完成复制所需的所有的蛋白质与其它因完成复制所需的所有的蛋白质与其它因子子DNA replicationPut DNA and protein together and ask replication question小的动物病毒分子：猿猴病毒小的动物病毒分子：猿猴病毒 SV40 (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork)

21、but not for initiation. 2. 酵母酵母Yeast (Saccharomyces cerevisiae): 1.4 X 107 bp in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system3. 无细胞提取物无细胞提取物Cell-free extract ：非洲爪蟾卵细胞：非洲爪蟾卵细胞提取物或者小麦胚芽提取物提取物或者小麦胚芽提取物. E3-2: 多复制子的起始多复制子的起始（Initiation of multiple rep

22、licons）DNA replication Timing Order20-50 复制子族复制子族 在细胞周期在细胞周期 S期同步进行期同步进行早早S-phase: 常染色质的复制常染色质的复制 （euchromatin replication）晚晚 S-phase: 异染色质复制异染色质复制 （heterochromatin replication）最后：着丝粒与端粒复制最后：着丝粒与端粒复制 （Centromeric and telomeric DNA replication ）2.准许因子（准许因子（Licensing factor） : Only initiate once per c

23、ell cycle 起始要求，用后失活（起始要求，用后失活（required for initiation and inactivated after use） 有丝分裂核膜裂解时才能进入胞核（有丝分裂核膜裂解时才能进入胞核（Can only enter into nucleus when the nuclear envelope dissolves at mitosis）Licensing factorInitiationInitiation: origin自主复制序列自主复制序列ARSs: autonomously replicating sequences Individual yeas

24、t replication origins (ARS) have been cloned into prokaryotic plasmids which allow these plasmids to replicate in yeast (a eukaryote). Minimal sequence: 11 bp A/TTTTATA/GTTTA/T (TATA box)起始点识别复合体起始点识别复合体ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for

25、 replication. 准许因子（准许因子（Licensing factor）E3-3: 复制叉与延伸复制叉与延伸 Replication fork & elongationDNA replication unwinding enzymesReplication forkUnwinding DNA from nucleosomes: 50 bp/sec, need helicases and replication protein A (RP-A) New nucleosomes are assembled to DNA from a mixture of old and newl

26、y synthesized histones after the fork passes.Elongation: 三类三类 DNA聚合酶聚合酶 参与参与.DNA pol a a: 引发酶的活性合成引发酶的活性合成RNA，接着延伸，接着延伸DNA，但很快被其它两个酶取代。，但很快被其它两个酶取代。DNA pol d d: 前导链前导链leading strand取代取代DNA pol a a. 合成长的合成长的DNA1. DNA pol e e: 后随链后随链lagging strand取代取代DNA pol a. 合成长的合成长的DNA. .合成的冈崎片段（合成的冈崎片段（ Okazaki f

27、ragments）非常短）非常短 (135 bp in SV40), 反应了来自反应了来自每个核小体的每个核小体的DNA的解旋的数量的解旋的数量. E3-4: 核基质核基质Nuclear MatrixDNA replication 由不溶性的蛋白纤维组成的支架，作为核中由不溶性的蛋白纤维组成的支架，作为核中各种反应进行时的组织架构行使功能，包括各种反应进行时的组织架构行使功能，包括DNA的复制与转录的复制与转录 A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear

28、 processing, including DNA replication, transcription复制工厂（复制工厂（Replication factories）: all the replication enzymes, DNA associated with the replication forks in replication 溴化氧尿苷溴化氧尿苷BUdR 标记标记 DNA免疫荧光法观察免疫荧光法观察 E3-3: 端粒复制端粒复制 Telomere replicationDNA replicationSolving the problem of Chromosomal ends

29、 shortening: 5-endsSemi-discontinuous replicationLigation DNA replicationOkazaki fragments冈崎片段冈崎片段telomeraseDNA polymerase 控制控制DNADNA复制的忠实性复制的忠实性校正（校正（Proofreading）：）： refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after

30、they have been added to the chainProcessive DNA polymerases have 35 exonuclease activitySupplemental 1byE. coli polymerase ProofreadingSupplemental 2Crystal structure of phage T7 DNA polymeraseExonuclease domaintemplate3.8. DNA 复复 制制 速速 率率 及及 拷拷 贝贝 数数 的的 调调 控控 ( ? ! )( ? ! ) a) 影响因素影响因素 多种专一性的蛋白质因子浓

31、度多种专一性的蛋白质因子浓度除引物以外的其他除引物以外的其他RNARNA分子（分子（anti-RNAanti-RNA）营养条件（营养条件（aa starvedaa starved的抑制的抑制) )（原核生物）（原核生物）细胞复制周期速率的影响（真核生物）细胞复制周期速率的影响（真核生物）b) 严格的自我调控机制严格的自我调控机制 e.g. plasmid （parasite） independent replication 严谨型严谨型stringent type (1-2copies) 松弛型松弛型relaxed type (10-100copies) RopRop蛋白蛋白 反义反义RNA1

32、 RNA-2 positive control RNA-2 positive control 正调控 0-555 0 RNA-2 RNaseHOH RNA-2复制的调控复制的调控e.g. ColEI plasmide.g. ColEI plasmid负调控负调控DNA复制方向RNA-1110 bp RNase H 不能识别不能识别D.S. RNA-1/RNA-2, 不能形成不能形成primer 的3-OH -555 -4450RNA-2RNA-1RNA-2 110 bp D.S. RNARNA-1 0 When RNA-2200-360Nt Rop Rop gene expressiongene expression RNA-1RNA-1 / / RNA-2 D.S. repressionRNA-2 D.S. repression RNA-1 / RNA-2 不能形成不能形成primer的的