《分子生物学》3 chapter8.ppt
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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
4、-discontinuously 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 synthe
5、sis,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 subu
6、nits 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 pr
7、imer: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 pr
8、ocess 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,Catalyt
9、ic 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 templa
10、te 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 a
11、ssociation 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
12、 specialization 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 a
13、verage 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 dra
14、matically 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
15、of 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 Replicati
16、on 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 rem
17、oves 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 (SS
18、Bs) 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 superc
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