分子生物學(xué)教學(xué)課件：CHAPTER 8 The replication of DNA

1、回顧前一次課所學(xué)的內(nèi)容回顧前一次課所學(xué)的內(nèi)容1. 染色體、染色質(zhì)和核小體以及染色體的結(jié)構(gòu)特點(diǎn)。染色體、染色質(zhì)和核小體以及染色體的結(jié)構(gòu)特點(diǎn)。2. DNA包裝成染色體的重要性是什么？包裝成染色體的重要性是什么？3. 基因組大小、基因數(shù)量和基因密度的概念。基因組大小、基因數(shù)量和基因密度的概念。4. 在真核細(xì)胞中，影響基因密度大小的因素什么？在真核細(xì)胞中，影響基因密度大小的因素什么？5. DNA重復(fù)序列的特點(diǎn)和分布。重復(fù)序列的特點(diǎn)和分布。6. 染色體中的哪些元件不參與基因的表達(dá)？染色體中的哪些元件不參與基因的表達(dá)？7. 什么是細(xì)胞周期？真核染色體在細(xì)胞周期中的復(fù)制和分裂過(guò)程。什么是細(xì)胞周期？真核染色體

2、在細(xì)胞周期中的復(fù)制和分裂過(guò)程。8. 端粒酶的功能的是什么？端粒酶的功能的是什么？9. 細(xì)胞周期檢查點(diǎn)的作用是什么？細(xì)胞周期檢查點(diǎn)的作用是什么？10. 核小體的組成，組蛋白的結(jié)構(gòu)特點(diǎn)。核小體的組成，組蛋白的結(jié)構(gòu)特點(diǎn)。12. 能夠調(diào)節(jié)染色質(zhì)結(jié)構(gòu)的因素有哪些？能夠調(diào)節(jié)染色質(zhì)結(jié)構(gòu)的因素有哪些？ CHAPTER 8:The replication of DNAThe Chemistry of DNA SynthesisCHAPTER 8CHAPTER 8 In 1957, M. Meselson and F. W. Stahl successfully obtained the experimental

3、 proof for the semi-conservative replication of DNA. They did this by inventing a new technique called Density Gradient Centrifugation. Their paper was published in 1958 and ever since, the experiment has often been referred to as: one of the most beautiful experiments in biology. How It Began:CHAPT

4、ER 8CHAPTER 8Meselson and Stahl Experiment Meselson及及Stahl：提出半保留模型：提出半保留模型M. Meselson and F.W. Stahl. 1958. The replication of DNA in E. coli. Proc. Natl. Acad. Sci. U.S.A. 44: 671-682. semi conservation replicationCHAPTER 8CHAPTER 8Meselson and Stahl.swfCHAPTER 8CHAPTER 8CsCl (氯化銫氯化銫)密度梯密度梯度離心分離度離心

5、分離DNASemi-ConservationReplicationComplications of DNA replication Enzymes The Topological Problem Direction problem: semi-discontinuously PrimingCHAPTER 8CHAPTER 8T1: The Chemistry of DNA SynthesisT2: The Mechanism of DNA PolymeraseT3: The Replication Fork T4: The Specialization of DNA Polymerases T

6、5: DNA Synthesis at the Replication Fork T6: Initiation of DNA ReplicationT7: Binding and UnwindingT8: Finishing ReplicationLecture TopicsGeneralDetailedCHAPTER 8CHAPTER 8The first part describes the basic chemistry of DNA synthesis and the function of the DNA polymerase.CHAPTER 8CHAPTER 8CHAPTER 8C

7、HAPTER 8T1: The Chemistry of DNA DNA synthesis requires deoxynucleoside triphosphates and a primer: template junction. DNA is synthesized by extending the 3 end of the primer. Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis.(1) DNA Synthesis Requires Deoxynucleoside Triphosp

8、hates and a Primer:Template junction CHAPTER 8CHAPTER 8Figure 8-1 Substrates required for DNA synthesis.dGTP. dCTP. dATP, or dTTPCHAPTER 8CHAPTER 8(2) DNA Is Synthesized by Extending the 3 End of the PrimerFigure 8-2 Diagram of the mechanism of DNA synthesis.5 3 DNA的結(jié)構(gòu)式的結(jié)構(gòu)式CHAPTER 8CHAPTER 8T2: The

9、Mechanism of DNA Polymerase1. DNA聚合酶以聚合酶以脫氧核苷酸三磷酸脫氧核苷酸三磷酸（dNTPs）為）為底物底物，沿模板的，沿模板的35方方向，將對(duì)應(yīng)的脫氧核苷酸連接到新生向，將對(duì)應(yīng)的脫氧核苷酸連接到新生DNA鏈的鏈的3端，使新生鏈沿端，使新生鏈沿53方向方向延長(zhǎng)。延長(zhǎng)。DNA Polymerase (Pol)2. 新鏈與原有的模板鏈新鏈與原有的模板鏈序列序列互補(bǔ)，亦與互補(bǔ)，亦與模板鏈的原配對(duì)鏈序列一致。模板鏈的原配對(duì)鏈序列一致。3. DNA聚合酶均以聚合酶均以53方向合成方向合成DNA，且均不能且均不能“重新重新”（de novo）合成）合成DNA，而只能將脫氧

10、核苷酸加到已有的，而只能將脫氧核苷酸加到已有的RNA或或DNA的的3端端羥基羥基上。上。 (1) DNA Pol use a single active site to catalyze DNA synthesis A single site to catalyze the addition of any of the four dNTPs. Recognition of different dNTP by monitoring the ability of incoming dNTP in forming A-T and G-C base pairs; incorrect base pair

11、 dramatically lowers the rate of catalysis. Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site.CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8Figure 8-3 Correctly paired bases are required for DNA-Pol-catalyzed nucleotide addition.CHAPTER 8CHAPTER 8Figure 8-4 Schematic illustra

12、tion of the steric constraints preventing DNA Pol from using rNTP precurson.Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site.In DNA Pol, the nucleotide binding pocket is too small to allow the presence of a 2-0H on the incoming nucleotide. This space is occupied by

13、 two amino acids (discriminator amino acids辨識(shí)氨基酸辨識(shí)氨基酸) that make van der Waals contacts with the sugar ring.CHAPTER 8CHAPTER 8(2) DNA Pol Resemble a That Grips the Primer:Template Junction Schematic of DNA Pol bound to a primer: template junctionA similar view of the T7 DNA pol bound to DNAFigure 8-

14、5 Three-dimensional structure of DNA Pol resembles a right hand.CHAPTER 8CHAPTER 8Two metal ions bound to DNA polymerase catalyze nucleotide addition.DNA Pol “grips” the template and the incoming nucleotide when a correct base pair is made.Figure 8-6Figure 8-7CHAPTER 8CHAPTER 8Figure 8-8 Illustratio

15、n of the path of the template DNA through the DNA Pol.ThumbFingersPalmCHAPTER 8CHAPTER 8手掌域：手掌域：1）包括兩個(gè)催化位點(diǎn)：一個(gè)是為加入）包括兩個(gè)催化位點(diǎn)：一個(gè)是為加入dNTP的，而另一個(gè)是為的，而另一個(gè)是為去除錯(cuò)配的去除錯(cuò)配的dNTP。2）聚合位點(diǎn)有催化的基本元件，可結(jié)合）聚合位點(diǎn)有催化的基本元件，可結(jié)合2個(gè)二價(jià)金屬離子，從個(gè)二價(jià)金屬離子，從而改變正確堿基配對(duì)的而改變正確堿基配對(duì)的dNTP和引物和引物3-OH周圍的化學(xué)環(huán)境。周圍的化學(xué)環(huán)境。3）除了催化作用外，手掌域還負(fù)責(zé)檢查最新加入的核苷酸堿）除了催化

16、作用外，手掌域還負(fù)責(zé)檢查最新加入的核苷酸堿基配對(duì)的準(zhǔn)確性。聚合酶的這一區(qū)域與新合成的基配對(duì)的準(zhǔn)確性。聚合酶的這一區(qū)域與新合成的DNA小溝小溝中的堿基對(duì)形成大量的氫鍵。中的堿基對(duì)形成大量的氫鍵。 DNA Pol-Palm DomainCHAPTER 8CHAPTER 8 手指域：對(duì)催化也很重要。手指域：對(duì)催化也很重要。 1）手指域中的幾個(gè)殘基可與引入的）手指域中的幾個(gè)殘基可與引入的dNTP結(jié)合，結(jié)合，一旦引入的一旦引入的dNTP與模板之間形成正確的堿基配與模板之間形成正確的堿基配對(duì)，手指域即發(fā)生移動(dòng)，包圍住對(duì)，手指域即發(fā)生移動(dòng)，包圍住dNTP。這種閉。這種閉合形式使引入的核苷酸與催化的金屬離子密

17、切接合形式使引入的核苷酸與催化的金屬離子密切接觸，從而促進(jìn)催化反應(yīng)。觸，從而促進(jìn)催化反應(yīng)。 2）手指域的移動(dòng)使模板的磷酸二酯鍵骨架在活性）手指域的移動(dòng)使模板的磷酸二酯鍵骨架在活性部位后立即產(chǎn)生彎曲，使催化位點(diǎn)上引物后第一部位后立即產(chǎn)生彎曲，使催化位點(diǎn)上引物后第一個(gè)模板堿基暴露，便于與下一個(gè)配對(duì)堿基結(jié)合。個(gè)模板堿基暴露，便于與下一個(gè)配對(duì)堿基結(jié)合。 .DNA Polymerase-finger domainCHAPTER 8CHAPTER 8 手拇指域與催化的關(guān)聯(lián)不大，而是與最新合成手拇指域與催化的關(guān)聯(lián)不大，而是與最新合成的的DNA相互作用。從而：相互作用。從而：1）維持引物以及活性部位的正確位置

18、；）維持引物以及活性部位的正確位置；2）幫助維持）幫助維持DNA集合酶與其底物之間的緊密連接。集合酶與其底物之間的緊密連接。 DNA Polymerase-thumb domainCHAPTER 8CHAPTER 8ThumbFingersPalm維持引物以及活性部位的維持引物以及活性部位的正確位置；正確位置；幫助維持幫助維持DNA聚合酶與其聚合酶與其底物之間的緊密連接。底物之間的緊密連接。一旦引入的一旦引入的dNTP與模板之與模板之間形成正確的堿基配對(duì)，手間形成正確的堿基配對(duì)，手指域即發(fā)生閉合式移動(dòng)，從指域即發(fā)生閉合式移動(dòng)，從而包圍住而包圍住dNTP，使引入的，使引入的核苷酸與催化的金屬離子

19、密核苷酸與催化的金屬離子密切接觸，促進(jìn)催化反應(yīng)；另切接觸，促進(jìn)催化反應(yīng)；另外，手指域的移動(dòng)使模板的外，手指域的移動(dòng)使模板的磷酸二酯鍵骨架在活性部位磷酸二酯鍵骨架在活性部位后立即產(chǎn)生彎曲，使催化位后立即產(chǎn)生彎曲，使催化位點(diǎn)上引物后第一個(gè)模板堿基點(diǎn)上引物后第一個(gè)模板堿基暴露，便于與下一個(gè)配對(duì)堿暴露，便于與下一個(gè)配對(duì)堿基結(jié)合。基結(jié)合。手掌域不僅可以催化手掌域不僅可以催化dNTP的添加反應(yīng)，還可以去除錯(cuò)配的的添加反應(yīng)，還可以去除錯(cuò)配的dNTP；手掌域可以；手掌域可以結(jié)合結(jié)合2個(gè)二價(jià)金屬離子，從而改變正確堿基配對(duì)的個(gè)二價(jià)金屬離子，從而改變正確堿基配對(duì)的dNTP和引物和引物3-OH周圍的化周圍的化學(xué)環(huán)境；

20、手掌域還負(fù)責(zé)檢查最新加入的核苷酸堿基配對(duì)的準(zhǔn)確性。學(xué)環(huán)境；手掌域還負(fù)責(zé)檢查最新加入的核苷酸堿基配對(duì)的準(zhǔn)確性。(3) DNA Pol are processive enzymes DNA聚合酶是一種延伸酶。聚合酶是一種延伸酶。 1）延伸能力是酶處理多聚體底物時(shí)的一種特性。）延伸能力是酶處理多聚體底物時(shí)的一種特性。 2）DNA聚合酶的延伸能力定義為每次酶與引物模板接頭聚合酶的延伸能力定義為每次酶與引物模板接頭結(jié)合時(shí)所添加核苷酸的平均數(shù)。每個(gè)結(jié)合時(shí)所添加核苷酸的平均數(shù)。每個(gè)DNA聚合酶都有其聚合酶都有其特征性的延伸能力，范圍從每次結(jié)合時(shí)的幾個(gè)到特征性的延伸能力，范圍從每次結(jié)合時(shí)的幾個(gè)到5000多多個(gè)

21、堿基。個(gè)堿基。 DNA合成的速率與聚合酶的延伸能力密切相關(guān)。因?yàn)?，合成的速率與聚合酶的延伸能力密切相關(guān)。因?yàn)?，聚合酶與引物聚合酶與引物-模板接頭的最初結(jié)合是限速步驟。模板接頭的最初結(jié)合是限速步驟。 CHAPTER 8CHAPTER 8The thumb helps to maintain a strong association between the DNA polymerase and its substrate.DNA polymerases are processive enzymes; thus the rate of DNA synthesis is dramatically in

22、creased (1000 bp/sec).CHAPTER 8CHAPTER 8Figure 8-9聚合酶與引物聚合酶與引物-模板模板接頭的最初結(jié)合是接頭的最初結(jié)合是限速步驟（限速步驟（1sec)(4) Exonucleases proofread newly synthesized DNA The occasional flicking of the bases into “wrong” tautomeric form results in incorrect base pair and mis-incorporation of dNTP. (10-5 mistake) The mismat

23、ched dNTP is removed by proofreading exonuclease, a part of the DNA polymerase.How does the exonucleases work? Kinetic selectivityCHAPTER 8CHAPTER 8Figure 8-10CHAPTER 8CHAPTER 8 Proofreading exonucleases removes bases from the 3 end of mismatched DNA. proofreading_function.swfT1: The Chemistry of DN

24、A SynthesisT2: The Mechanism of DNA PolymeraseT3: The Replication Fork T4: The Specialization of DNA Polymerases T5: DNA Synthesis at the Replication Fork T6: Initiation of DNA ReplicationT7: Binding and UnwindingT8: Finishing ReplicationLecture TopicsGeneralDetailedCHAPTER 8CHAPTER 8The second part

25、 describes how the synthesis of DNA occurs in the context of an intact chromosome at replication forks. CHAPTER 8CHAPTER 8T3: The replication fork The junction between the newly separated template strands and the unreplicated duplex DNA. CHAPTER 8CHAPTER 8新分開(kāi)的模板鏈與未復(fù)制的雙鏈新分開(kāi)的模板鏈與未復(fù)制的雙鏈DNA之間的連接區(qū)稱為復(fù)制叉。之

26、間的連接區(qū)稱為復(fù)制叉。CHAPTER 8CHAPTER 8(1) Both strands of DNA are synthesized together at the replication fork.Figure 8-11Leading strand(前導(dǎo)鏈前導(dǎo)鏈)Lagging strand(后隨鏈后隨鏈)Okazaki fragment(1001000bp)Replication fork(2) DNA helicases unwind the double helix in advance of the replication forkCHAPTER 8CHAPTER 8Figure

27、 8-13DNA解旋酶分離雙螺旋的兩條鏈。DNA解旋酶具有5-3極性。CHAPTER 8CHAPTER 8(3) Single-stranded binding proteins (SSBs) stabilize single-stranded DNA Cooperative binding Sequence-independent manner(electrostatic interactions) Figure 8-15單鏈單鏈DNA結(jié)合蛋白結(jié)合蛋白(4) The initiation of a new strand of DNA require an RNA primer Primase

28、 is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA sequence. DNA Pol can extend both RNA and DNA primers annealed to DNA template CHAPTER 8CHAPTER 8(5) RNA primers must be removed to complete DNA replication A joint efforts of RNa

29、se H, DNA Pol & DNA ligase Figure 8-12 Removal of RNA promers from newly synthesized DNA.CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8(6) Topoisomerase removes supercoils produced by DNA unwinding at the replication forkFigure 8-16CHAPTER 8CHAPTER 8(7) Replication fork enzymes extend the range of DNA pol

30、ymerase DNA Pol can not accomplish replication without the help of other enzymes T4: The Specilization of DNA PolymerasesCHAPTER 8CHAPTER 8 Each organism has a distinct set of different DNA Pols Different organisms have different DNA PolsDNA Pol III holoenzyme: a protein complex responsible for E. c

31、oli genome replicationDNA Pol I: removes RNA primers in E. coli Eukaryotic cells have multiple DNA Pol. Three are essential to duplicate the genome: DNA Pol d d, DNA Pol e e and DNA Pol a a/primase. (What are their functions?) Pol switching in Eukaryotes: the process of replacing DNA Pol a a/primase

32、 with DNA Pol d d or DNA Pol e e. CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8(1) DNA polymerases are specialized for different roles in the cellCHAPTER 8CHAPTER 8DNA Pols of eukaryotesFigure 8-17 DNA polymerase switching during eukaryotic DNA replicationCHAPTER 8CHAPTER 8 the process of replacing DNA Pola

33、a/primase with DNA Pold d or DNA Pole e. 50100bp10010000bpCHAPTER 8CHAPTER 8(2) Sliding clamps dramatically increase DNA Pol processivity Encircle the newly synthesized double-stranded DNA and the Pol associated with the primer: template junction. Ensures the rapid rebinding of DNA Pol to the same p

34、rimer: template junction, and thus increases the processivity of Pol. Eukaryotic sliding DNA clamp is PCNACHAPTER 8CHAPTER 8Figure 8-18 Structure of a sliding DNA clamp.Figure 8-19 Sliding DNA Clamps Increase the Processivity of associated DNA Pols.CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8Figure 8-20 Thr

35、ee-dimensional structure of sliding DNA clamps isolated from different organism.Sliding DNA clamps are found across all organism and share a similar structuretwo copies of b proteintrimer of the PCNA proteintrimer of the gp45 proteinE.coli:EukaryoticT4 phageCHAPTER 8CHAPTER 8(3) Sliding clamps are o

36、pened and placed on DNA by clamp loaders Clamp loader is a special class of protein complex catalyzes the opening and placement of sliding clamps on the DNA, such a process occurs anytime a primer-template junction is present. Sliding clamps are only removed from the DNA once all the associated enzy

37、mes complete their function.CHAPTER 8CHAPTER 8ATP control of sliding DNA clamp loadingSliding clamp loaderCHAPTER 8CHAPTER 8 At the replication, the leading strand and lagging strand are synthesized simultaneously. To coordinate the replication of both strands, multiple DNA Pols function at the repl

38、ication fork. DNA Pol III holoenzyme is such an example.T5: DNA synthesis at the replication forkCHAPTER 8CHAPTER 8Composition of the DNA Pol III holoenzymeFigure 8-21CHAPTER 8CHAPTER 8Figure 8-22* Trombone modelCHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8dna_replication.swfInteractions be

39、tween replication fork proteins form the E. coli replisome Replisome is established by protein-protein interactions1. DNA helicase & DNA Pol III holoenzyme, this interaction is mediated by the clamp loader and stimulates the activity of the helicase (10-fold)2. DNA helicase & primase, which

40、is relatively week and strongly stimulates the primase function (1000-fold). This interaction is important for regulation the length of Okazaki fragments.CHAPTER 8CHAPTER 8 DNA Pol III holoenzyme, helicase and primase interact with each other to form replisome, a finely tuned factory for DNA synthes

41、is with the activity of each protein is highly coordinated.CHAPTER 8CHAPTER 8Interactions between replication fork proteins from the E. coli replisomeCHAPTER 8CHAPTER 8Figure 8-23 Binding of the DNA helicase to DNA pol III holoenzyme stimulates the rate of DNA strand separation.T1: The Chemistry of

42、DNA SynthesisT2: The Mechanism of DNA PolymeraseT3: The Replication Fork T4: The Specialization of DNA Polymerases T5: DNA Synthesis at the Replication Fork T6: Initiation of DNA ReplicationT7: Binding and UnwindingT8: Finishing ReplicationLecture TopicsGeneralDetailedCHAPTER 8CHAPTER 8The third par

43、t focuses on the initiation and termination of DNA replication. Note that DNA replication is tightly controlled in all cells and initiation is the step for regulation. CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8T6: Initiation of DNA replication(1) Specific genomic DNA sequences direct the initiation of DNA

44、 replication Origins of replication, the sites at which DNA unwinding and initiation of replication occur. 思考下列概念之間的關(guān)系：思考下列概念之間的關(guān)系： 復(fù)制子（復(fù)制子（replicon） 復(fù)制器（復(fù)制器（replicator） 復(fù)制起始位點(diǎn)（復(fù)制起始位點(diǎn)（ origins of replication ） 起始子（起始子（initiator） 起始位點(diǎn)識(shí)別復(fù)合體（起始位點(diǎn)識(shí)別復(fù)合體（origin recognition complex,ORC）Replicator: the enti

45、re site of cis-acting DNA sequences sufficient to direct the initiation of DNA replicationInitiator protein: specifically recognizes a DNA element in the replicator and activates the initiation of replicationFigure 8-24 the replication model.CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8The identification of

46、origins of replicationBox 8-5 Figure 1 Genetic identification of replicators (origins)CHAPTER 8CHAPTER 8(2) Replicator sequences include initiator binding sites and easily unwound DNAFigure 8-25 Structure of replication.起始子結(jié)合位點(diǎn)：綠色起始子結(jié)合位點(diǎn)：綠色1）含有起始子蛋白質(zhì)結(jié)合的位點(diǎn)，此位點(diǎn)是組裝復(fù)制起始機(jī)器的核心；2）含有一段富含AT的DNA，此段DNA容易解旋但并不自

47、發(fā)進(jìn)行。在復(fù)制器上，DNA的解旋是由復(fù)制起始蛋白控制的，這些蛋白被嚴(yán)格調(diào)控。如在E.Coli中，復(fù)制器為“oriC”。9核苷酸單位的基序是起始子DnaA的結(jié)合位點(diǎn)，在oriC”上重復(fù)5次，13核苷酸單位的基序重復(fù)3次，是起始時(shí)單鏈DNA形成的起始位點(diǎn)。 促進(jìn)促進(jìn)DNA解旋的元件：藍(lán)色解旋的元件：藍(lán)色第一段第一段DNA合成的位點(diǎn)：紅色合成的位點(diǎn)：紅色T7: Binding and Unwinding: origin selection and activation by the initiator proteinCHAPTER 8CHAPTER 8 Three different functio

48、ns of initiator protein: (1) binds to replicator, (2) distorts/unwinds a region of DNA, (3) interacts with and recruits additional replication factors DnaA in E. coli (all 3 functions), origin recognition complex (ORC) in eukaryotes (functions 1 & 3)Figure 8-26 Functions of the initiator protein

49、s during the initiation of DNA replication.CHAPTER 8CHAPTER 8CHAPTER 8CHAPTER 8(1) Protein-protein and protein-DNA interactions direct the initiation process DnaA recruits the DNA helicase, DnaB and the helicase loader DnaC DnaB interacts with primase to initiate RNA primer synthesis, see replisome

50、part for more details.Figure 8-27 Models for E. coli initiation of DNA replicationCHAPTER 8CHAPTER 8Figure 8-27 Models for E. coli initiation of DNA replicationCHAPTER 8CHAPTER 8下列概念之間的關(guān)系：下列概念之間的關(guān)系： 復(fù)制子（復(fù)制子（replicon） 復(fù)制器（復(fù)制器（replicator） 復(fù)制起始位點(diǎn)（復(fù)制起始位點(diǎn)（ origin of replication ） 起始子（起始子（initiator） 起始位點(diǎn)識(shí)

51、別復(fù)合體（起始位點(diǎn)識(shí)別復(fù)合體（origin recognition complex,ORC）(2) Eukaryotic chromosome are replicated exactly once per cell cycle, which is critical for these organimsCHAPTER 8CHAPTER 81）當(dāng)）當(dāng)DNA復(fù)制時(shí)，必須復(fù)制時(shí)，必須激活足夠多的起始位點(diǎn)，激活足夠多的起始位點(diǎn)，以保證每個(gè)以保證每個(gè)S期中每條染期中每條染色體都被完全復(fù)制；色體都被完全復(fù)制；2）通常一個(gè)細(xì)胞周期，起始通常一個(gè)細(xì)胞周期，起始位點(diǎn)只被激活一次，位點(diǎn)只被激活一次， 即即復(fù)制只完

52、成一次。如果某復(fù)制只完成一次。如果某些區(qū)域沒(méi)有被復(fù)制，就會(huì)些區(qū)域沒(méi)有被復(fù)制，就會(huì)產(chǎn)生染色體斷裂。產(chǎn)生染色體斷裂。 Figure 8-29 Replicators are inactivated by DNA replicationCHAPTER 8CHAPTER 8(3) Pre-replicative complex (pre-RC) formation directs the initiation of replication in eukaryotes Initiation in eukaryotes requires two distinct steps Replicator sele

53、ction: the process of identifying sequences for replication initiation (G1 phase), which is mediated by the formation of pre-RCs at the replicator region. CHAPTER 8CHAPTER 8Figure 8-30 Steps in the formation of the prerelicative complex (pre-RC)CHAPTER 8CHAPTER 8AAA+AAA+pre-RC的形成并不導(dǎo)致起始的形成并不導(dǎo)致起始位點(diǎn)位點(diǎn)D

54、NA立即被解旋或者立即被解旋或者DNA聚合酶的募集，而是只聚合酶的募集，而是只有在細(xì)胞從細(xì)胞周期的有在細(xì)胞從細(xì)胞周期的G1到到達(dá)達(dá)S期后，期后，G1期形成的期形成的pre-RC才被激活，并啟動(dòng)復(fù)制才被激活，并啟動(dòng)復(fù)制起始。起始。 Origin activation: pre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only when the cells enter S phase.CHAPTER 8CHAPTER 8Kinases（激酶）：是一類可以將磷酸基團(tuán)共價(jià)連接到靶蛋激酶）：是一類可以將

55、磷酸基團(tuán)共價(jià)連接到靶蛋白上的蛋白質(zhì)。所有的激酶在白上的蛋白質(zhì)。所有的激酶在G1期失活，進(jìn)入期失活，進(jìn)入S期后被激活。期后被激活。 Assembly of the eukaryotic replication forkCHAPTER 8CHAPTER 8Figure 8-31pre-RC(4) Pre-RC formation and activation is regulated to allow only a single round of replication during each cell cycle.Only one opportunity for pre-RCs to form,

56、 and only one opportunity for pre-RC activation.CHAPTER 8CHAPTER 8Cdks: cyclin-dependent kinasesFigure 8-32 Effect of Cdk activity on pre-RC formation and activationCHAPTER 8CHAPTER 8Figure 8-32 Cell cycle regulation of Cdk activity and pre-RC formatinCHAPTER 8CHAPTER 8Similarities between eukaryoti

57、c and prokaryotic DNA replication initiationCHAPTER 8CHAPTER 8Same points: 1) Recognize the replicator; 2) Assembles the DNA helicase on the replicator; 3) Helicase generates a region of ssDNA for RNA primer synthesis; 4) The replisome assembles and start DNA replication.Different points: In bacteri

58、a cells 1) initiate replication more than once per cell cycle; 2) focus regulation on the binding of the DnaA initiator protein to the DNA . E. Coli DNA replication is regulated by DnaA-ATP levels and SeqACHAPTER 8CHAPTER 8T8: FINISHING REPLICATIONCHAPTER 8CHAPTER 8 DNA 復(fù)制的完成需要一系列復(fù)雜的過(guò)程，復(fù)制的完成需要一系列復(fù)雜的

59、過(guò)程，對(duì)于環(huán)形和線性染色體來(lái)說(shuō)，這些過(guò)程有所對(duì)于環(huán)形和線性染色體來(lái)說(shuō)，這些過(guò)程有所不同。不同。 環(huán)形染色體的復(fù)制叉機(jī)器能夠復(fù)制整個(gè)分環(huán)形染色體的復(fù)制叉機(jī)器能夠復(fù)制整個(gè)分子，但是產(chǎn)生的子代分子之間是相互拓?fù)溥B子，但是產(chǎn)生的子代分子之間是相互拓?fù)溥B接的。接的。 線性染色體最末端處的復(fù)制是通過(guò)端粒酶線性染色體最末端處的復(fù)制是通過(guò)端粒酶延伸染色體延伸染色體3端來(lái)解決末端復(fù)制的問(wèn)題。端來(lái)解決末端復(fù)制的問(wèn)題。(1) Type II topoisomerases are required to separate daughter DNA moleculesCHAPTER 8CHAPTER 8(2) Lagg

60、ing-strand synthesis is unable to copy the extreme ends of linear chromosomesCHAPTER 8CHAPTER 8End replication problemFigure 8-36 Protein priming as a solution to the end replication problem.CHAPTER 8CHAPTER 8TelomereStructure of human telomereCHAPTER 8CHAPTER 8 端粒是染色體末端的一種特殊結(jié)構(gòu)，是端粒是染色體末端的一種特殊結(jié)構(gòu)，是DNA與相關(guān)蛋白質(zhì)的復(fù)合與相關(guān)蛋白質(zhì)的復(fù)合體。端粒