《分子生物学》3chaVIP

1、CHAPTER 8:,The replication of DNA,Meselson and Stahls Experiment,M. Meselson and F.W. Stahl. 1958. The replication of DNA in Escherichia coli Proc. Natl. Acad. Sci. U.S.A. 44: 671-682.,M. Meselson and F. W. Stahl were interested in trying to devise a way to prove or disprove Watson and Cricks model

2、of semi-conservative replication. In 1957, they successfully obtained the experimental 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 bee

3、n referred to as: one of the most beautiful experiments in biology.,How It Began:,CsCl 密度梯度离心分离DNA,A. The Meselson-Stall experiment,B. the interpretation,(CsCl gradient centrifuge),Semi-ConservationReplication,Complications of DNA replication,Enzymes The Topological Problem Direction problem: semi-d

4、iscontinuously Priming,The Chemistry of DNA Synthesis DNA Polymerase The Replication Fork The Replication Process,CHAPTER 8 The replication of DNA,Raw materials Reaction direction Chemical reaction The driving force for DNA synthesis,The Chemistry of DNA Synthesis,Substrate required for DNA synthesi

5、s,Diagram of the mechanism of DNA synthesis,The Chemistry of DNA Synthesis DNA Polymerase The Replication Fork The Replication Process,CHAPTER 8 The replication of DNA,DNA Polymerase,The specialization of DNA polymerases,The mechanism of DNA Polymerase,kinds of DNA polymerases The role of the subuni

6、ts of DNA polymerases,Function mechanism,DNA polymerases of bacteria,The composition of the DNA Pol III holoenzyme,Sliding clamps,Encircle the newly synthesized double-stranded DNA and the polymerase associated with the primer:template junction Ensures the rapid rebinding of DNA Pol to the same prim

7、er:template junction, and thus increases the processivity of Pol. Eukaryotic sliding DNA clamp is PCNA,Sliding DNA Clamp,Sliding DNA clamps are found across all organism and share a similar structure,Sliding DNA Clamp Increases Processivity,DNA polymerases of eukaryotes,Polymerase switching,the proc

8、ess of replacing DNA Pola/primase with DNA Pold or DNA Pole.,DNA Polymerase,The specialization of DNA polymerases,The mechanism of DNA Polymerase,Mechanism Function,The specialization of DNA polymerases,kinds of DNA polymerases The role of the subunits of DNA polymerases,Thumb,Fingers,Palm,Catalytic

9、 sites for addition and removal of dNTPs. Binds to two metal ions that alter the chemical environment around the catalytic site. Stabilization of the pyrophosphate,DNA Polymerase-palm domain,Binds to the incoming dNTP, encloses the correct paired dNTP to the position for catalysis Bends the template

10、 to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotide,DNA Polymerase-finger domain,Not directly involved in catalysis Interacts with the synthesized DNA to maintain correct position of the primer and the active site, and to maintain a strong ass

11、ociation between DNA Pol and its substrate.,DNA Polymerase-thumb domain,DNA Polymerase-palm domain,DNA Polymerase-finger domain,Thumb,Fingers,Palm,DNA Polymerase,The specialization of DNA polymerases,The mechanism of DNA Polymerase,Subunits of DNA polymerases The role of the subunits,Mechanism,The s

12、pecialization of DNA polymerases,Subunits of DNA polymerases The role of the subunits,Function,DNA rapid processive synthesis DNA accurate synthesis,DNA Pol are processive enzymes,Processivity is a characteristic of enzymes that operate on polymeric substrates. The processivity of DNA Pol is the ave

13、rage number of nucleotides added each time the enzyme binds a primer:template junction (a few50,000).,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 drama

14、tically lowers the rate of catalysis Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site.,DNA accurate synthesis,Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site,Exonucleases proofread newly synthesized DNA,The occasional flicking of

15、 the bases into “wrong” tautomeric form results in incorrect base pair and mis-incorporation of dNTP. (10-5 mistake) The mismatched dNMP is removed by proofreading exonuclease.,Post-replication mismatch repair process,The Chemistry of DNA Synthesis DNA Polymerase The Replication Fork The Replication

16、 Process,The replication fork,The junction between the newly separated template strands and the unreplicated duplex DNA,3. Priming,Reason: polymerase proofreading activity priming process:,1. Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNA Topoisomerase remov

17、es supercoils,2. Semi-discontinuously,Leading strand : Lagging strand:Okazaki fragment:,DNA helicases unwind the double helix in advance of the replication fork,Hexameric protein,Topoisomerase removes supercoils produced by DNA unwinding at the replication fork,Single-stranded binding proteins (SSBs

18、) stabilize single-stranded DNA,Cooperative binding Sequence-independent manner (electrostatic interactions),3. Priming,Reason: polymerase proofreading activity priming process:,1. Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNA Topoisomerase removes supercoi

19、ls,2. Semi-discontinuously,Leading strand : Lagging strand:Okazaki fragment:,Mechanism Of DNA Chain Growth, II. Accumulation Of Newly Synthesized Short Chains In E. Coli Infected With Ligase Defective T4 Phages* Kazunori Sugimoto, Tsuneko Okazaki, and Reiji Okazaki, Proc. Natl. Acad. Sci. U.S.A 1968

20、 60: 1356-1362.,Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968),Reiji Okazaki was born near Hiroshima, Japan, in 1930. He was a teenager there at the time of the explosion of the first of two nuclear bombs that the US dropped at the end of World War II. His

21、scientific career was cut short by his untimely death from cancer in 1975 at the age of 44, perhaps related to his exposure to the fallout of that blast.,Tsuneko Okazaki, until recently, was a professor at The University of Nagoya where she was the first woman at that institution to be named a profe

22、ssor. Currently she is on the faculty of Medicine in Fujita, and does research on centromeres.,Okazakis Experiment,This experiment clearly showed the existence of 1000-2000-nucleotide fragments called “Okazaki fragments”. These fragments later became incorporated into normal DNA strands, at the comp

23、letion of DNA replication. According to Okazakis research DNA replication follows a “back and fill” mechanism.,E.coli t-,2, 7, 15, 60,pulse-labeling in dT-H3,stop in KCN 0,D.S. DNA,S.S. DNA,Density gradient of sucrose,Measure H3-T,pulse-chase,20 in dT-H3 30,transfer to dT then continue,H3-T,pulse-ch

24、ase,pulse-labeling,120,60,15,7,2,DNA replication in Okazaki fragment 1kb,3. Priming,Reason: polymerase proofreading activity priming process:,1. Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNA Topoisomerase removes supercoils,2. Semi-discontinuously,Leading s

25、trand : Lagging strand:Okazaki fragment:,The initiation of a new strand of DNA require an RNA primer,Primase 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 t

26、emplate,Priming of DNA synthesis,RNA primers must be removed to complete DNA replication,A joint efforts of RNase H, DNA polymerase & DNA ligase,3. Priming,Reason: polymerase proofreading activity priming process:,1. Unwind the double helix,DNA helicases separate the two base-paired strands of dupie

27、x DNA Topoisomerase removes supercoils,2. Semi-discontinuously,Leading strand : Lagging strand:Okazaki fragment:,The Chemistry of DNA Synthesis DNA Polymerase The Replication Fork The Replication Process,CHAPTER 8 The replication of DNA,The Replication Process,Initiation Elongation Termination,Origi

28、ns of replication,the sites at which DNA unwinding and initiation of replication occur.,The replicon model of replication initiation,replicator initiator,Initiation of DNA replication,in E. coli In Eukaryote,The replicon model of replication initiation,3/18/05,Proposed by Jacob and Brenner in 1963 A

29、ll the DNA replicated from a particular origin is a replicon Two components, replicator and initiator, control the initiation of replication,Initiator protein: specifically recognizes a DNA element in the replicator and activates the initiation of replication,Replicator: the entire site of cis-actin

30、g DNA sequences sufficient to direct the initiation of DNA replication,Replicator sequences,3/18/05,OriC in E. coli chromosomal DNA,Three different functions of initiator protein:,binds to replicator, distorts/unwinds a region of DNA, interacts with and recruits additional replication factors,DnaA i

31、n E. coli (all 3 functions), origin recognition complex (ORC) in eukaryotes (functions 1 & 3),Initiation of DNA replication in E. coli,Initiation in E. coli,Origins of replication,the sites at which DNA unwinding and initiation of replication occur.,The replicon model of replication initiation,repli

32、cator initiator,Initiation of DNA replication,in E. coli In Eukaryote,Prereplicative Complex,pre-RC activation & assembly of the replication fork in eukaryotes,pre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only when the cells enter S phase.,Origin activation:,Pre-RC form

33、ation and activation is regulated to allow only a single round of replication during each cell cycle.,Only one opportunity for pre-RCs to form, and only one opportunity for pre-RC activation.,The regulation of initiation of replication,Figure 8-31 Effect of Cdk activity on pre-RC formation and activation,Cell cycle regulation of Cdk activity and pre-RC fo