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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 of semi-conse
2、rvative 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 been referred to as:o
3、ne 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,EnzymesThe Topological ProblemDirection problem:semi-discontinuouslyPriming,Th
4、e Chemistry of DNA SynthesisDNA PolymeraseThe Replication ForkThe Replication Process,CHAPTER 8 The replication of DNA,Raw materialsReaction direction Chemical reaction The driving force for DNA synthesis,The Chemistry of DNA Synthesis,Substrate required for DNA synthesis,Diagram of the mechanism of
5、 DNA synthesis,The Chemistry of DNA SynthesisDNA PolymeraseThe Replication ForkThe 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 subunits of DNA polymerases,Functionme
6、chanism,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 junctionEnsures the rapid rebinding of DNA Pol to the same primer:template junction,and thus incr
7、eases 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 process of replacing DNA Pola/primase wi
8、th DNA Pold or DNA Pole.,DNA Polymerase,The specialization of DNA polymerases,The mechanism of DNA Polymerase,MechanismFunction,The specialization of DNA polymerases,kinds of DNA polymerases The role of the subunits of DNA polymerases,Thumb,Fingers,Palm,Catalytic sites for addition and removal of dN
9、TPs.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 catalysisBends the template to expose the only nucleotide at the tem
10、plate that ready for forming base pair with the incoming nucleotide,DNA Polymerase-finger domain,Not directly involved in catalysisInteracts with the synthesized DNA to maintain correct position of the primer and the active site,and to maintain a strong association between DNA Pol and its substrate.
11、,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 specialization of DNA polymerases,Subunits o
12、f DNA polymerases The role of the subunits,Function,DNA rapid processive synthesisDNA 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 average number of nucleotides added each time th
13、e 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 dramatically lowers the rate of catalysis Distinguish
14、 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 the bases into“wrong”tautomeric form results in
15、 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 SynthesisDNA PolymeraseThe Replication ForkThe Replication Process,The replication fork,The junction between the
16、newly separated template strands and the unreplicated duplex DNA,3.Priming,Reason:polymerase proofreading activitypriming process:,1.Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNATopoisomerase removes supercoils,2.Semi-discontinuously,Leading strand:Lagging
17、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)stabilize single-stranded DNA,Cooperative bindingSequence-indep
18、endent manner(electrostatic interactions),3.Priming,Reason:polymerase proofreading activitypriming process:,1.Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNATopoisomerase removes supercoils,2.Semi-discontinuously,Leading strand:Lagging strand:Okazaki fragment
19、:,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,1968 60:1356-1362.,Evidence for the Semi-Discontinuous replication model was provided by the Okazakis(1968),Reiji O
20、kazaki 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 scientific career was cut short by his untimely death from cancer in 1975 at the age of 44,perhaps related to his exp
21、osure to the fallout of that blast.,Tsuneko Okazaki,until recently,was a professor atThe University of Nagoya where she was the first woman at that institution to be named a professor.Currently she is on the faculty of Medicine in Fujita,and does research on centromeres.,Okazakis Experiment,This exp
22、eriment clearly showed the existence of 1000-2000-nucleotide fragments called“Okazaki fragments”.These fragments later became incorporated into normal DNA strands,at the completion of DNA replication.According to Okazakis research DNA replication follows a“back and fill”mechanism.,E.coli t-,2,7,15,6
23、0,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-chase,pulse-labeling,120,60,15,7,2,DNA replication in Okazaki fragment 1kb,3.Priming,Reason:polymerase proofreading activitypriming proc
24、ess:,1.Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNATopoisomerase removes supercoils,2.Semi-discontinuously,Leading strand:Lagging strand:Okazaki fragment:,The initiation of a new strand of DNA require an RNA primer,Primase is a specialized RNA polymerase d
25、edicated 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,Priming of DNA synthesis,RNA primers must be removed to complete DNA replication,A joint efforts of RNase H,DNA polymerase&DNA ligase,3.
26、Priming,Reason:polymerase proofreading activitypriming process:,1.Unwind the double helix,DNA helicases separate the two base-paired strands of dupiex DNATopoisomerase removes supercoils,2.Semi-discontinuously,Leading strand:Lagging strand:Okazaki fragment:,The Chemistry of DNA SynthesisDNA Polymera
27、seThe Replication ForkThe Replication Process,CHAPTER 8 The replication of DNA,The Replication Process,InitiationElongation Termination,Origins of replication,the sites at which DNA unwinding and initiation of replication occur.,The replicon model of replication initiation,replicatorinitiator,Initia
28、tion of DNA replication,in E.coliIn Eukaryote,The replicon model of replication initiation,3/18/05,Proposed by Jacob and Brenner in 1963All the DNA replicated from a particular origin is a repliconTwo components,replicator and initiator,control the initiation of replication,Initiator protein:specifi
29、cally recognizes a DNA element in the replicator and activates the initiation of replication,Replicator:the entire site of cis-acting 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 initiato
30、r protein:,binds to replicator,distorts/unwinds a region of DNA,interacts with and recruits additional replication factors,DnaA in 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,t
31、he sites at which DNA unwinding and initiation of replication occur.,The replicon model of replication initiation,replicatorinitiator,Initiation of DNA replication,in E.coliIn Eukaryote,Prereplicative Complex,pre-RC activation&assembly of the replication fork in eukaryotes,pre-RCs are activated by t
32、wo protein kinases(Cdk and Ddk)that are active only when the cells enter S phase.,Origin activation:,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,and only one opportunity for pre-RC activatio
33、n.,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 formatin,The Replication Process,InitiationElongation Termination,At the replication,the leading strand and lagging strand are synthes
34、ized simultaneously.To coordinate the replication of both strands,multiple DNA Pols function at the replication fork.DNA Pol III holoenzyme is such an example.,The composition of the DNA Pol III holoenzyme,Trombone model,The Replication Process,InitiationElongation Termination,Type II topoisomerases
35、 are required to separate daughter DNA molecules,Telomeres cannot be maintained by semi-conservative DNA replication,3,5,5,3,3,5,5,3,Leading strand synthesis,Lagging strand synthesis,3,5,5,3,5,3,5,3,DNA pol,DNA pol,Region of unreplicated DNA,Leading strand synthesis,Lagging strand synthesis,Telomere
36、s cannot be maintained by semi-conservative DNA replication,3,5,5,3,5,3,5,3,Region of unreplicated DNA,Leading strand synthesis,Lagging strand synthesis,Telomeres cannot be maintained by semi-conservative DNA replication,Telomerase ReplicatesTelomeres,Telomerase Extends 3 End,The Chemistry of DNA SynthesisDNA PolymeraseThe Replication ForkThe Replication Process,
