《分子生物學(xué)》3 cha

1、How It Began:CsCl 密度梯度離心分離密度梯度離心分離DNA(CsCl gradient centrifuge) (CsCl gradient centrifuge) N15 N14N15 DNADNASemi-ConservationReplicationCHAPTER 8 The replication of DNAThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesisSubstrate required for DNA synthesisCHAPTER 8 The replication of DNAl Th

2、e mechanism of DNA Polymerasekinds of DNA polymerases The role of the subunits of DNA polymerases FunctionmechanismSliding DNA ClampSliding DNA clamps are found across all organism and share a similar structureDNA polymerases of eukaryotes the process of replacing DNA Pola/primase with DNA Pold or D

3、NA Pole. l The mechanism of DNA PolymeraseMechanismFunctionkinds of DNA polymerases The role of the subunits of DNA polymerasesThumbFingersPalmlCatalytic sites for addition and removal of dNTPs. lBinds to two metal ions that alter the chemical environment around the catalytic site. lStabilization of

4、 the pyrophosphateDNA Polymerase-palm domainlBinds to the incoming dNTP, encloses the correct paired dNTP to the position for catalysislBends the template to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotideDNA Polymerase-finger domainlNot direc

5、tly involved in catalysislInteracts 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.DNA Polymerase-thumb domainDNA Polymerase-finger domainThumbFingersPalml The mechanism of DNA Polymerase

6、Subunits of DNA polymerases The role of the subunitsMechanismSubunits of DNA polymerases The role of the subunitsFunctionDNA Pol are processive enzymes lProcessivity is a characteristic of enzymes that operate on polymeric substrates.lThe processivity of DNA Pol is the average number of nucleotides

7、added each time the enzyme binds a primer:template junction (a few50,000). lA single site to catalyze the addition of any of the four dNTPs. lRecognition 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 o

8、f catalysis lDistinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site.DNA accurate synthesisDistinguish between rNTP and dNTP by steric exclusion of rNTPs from the active siteExonucleases proofread newly synthesized DNA The occasional flicking of the bases into “wrong” tau

9、tomeric 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 process3. PrimingReason: polymerase proofreading activitypriming process:1. Unwind the double helix DNA helicases sepa

10、rate the two base-paired strands of dupiex DNA Topoisomerase removes supercoils 2. Semi-discontinuouslyLeading strand :Lagging strand:Okazaki fragment:DNA helicases unwind the double helix in advance of the replication forkHexameric proteinTopoisomerase removes supercoils produced by DNA unwinding a

11、t the replication forkSingle-stranded binding proteins (SSBs) stabilize single-stranded DNA Cooperative binding Sequence-independent manner (electrostatic interactions) 3. PrimingReason: polymerase proofreading activitypriming process:1. Unwind the double helix DNA helicases separate the two base-pa

12、ired strands of dupiex DNA Topoisomerase removes supercoils 2. Semi-discontinuouslyLeading strand :Lagging strand:Okazaki fragment: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 a

13、t 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 exposure to the fallout of that blast.Tsuneko Okazaki, until recently,

14、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. t- 2, 7, 15, 60pulse-labeling in dT-H3 stop in KCN 0stop in KCN 0D.S. DNA S.S. DNA Density gra

15、dient of sucrose Measure H3-T pulse-chase 20 in dT-H3 30 transfer to dT then transfer to dT then continuecontinueH3-T pulse-chase pulse-labeling 120 60 15 7 2 10S (1kb) 70S (Prok. 400Nt/sec) DNA replication in Okazaki fragment 1kb3. PrimingReason: polymerase proofreading activitypriming process:1. U

16、nwind the double helix DNA helicases separate the two base-paired strands of dupiex DNA Topoisomerase removes supercoils 2. Semi-discontinuouslyLeading strand :Lagging strand:Okazaki fragment:The initiation of a new strand of DNA require an RNA primer Primase is a specialized RNA polymerase dedicate

17、d 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 RNA primers must be removed to complete DNA replicationA joint efforts of RNase H, DNA polymerase & DNA ligase 3. PrimingReason: polymera

18、se proofreading activitypriming process:1. Unwind the double helix DNA helicases separate the two base-paired strands of dupiex DNA Topoisomerase removes supercoils 2. Semi-discontinuouslyLeading strand :Lagging strand:Okazaki fragment:CHAPTER 8 The replication of DNAlthe sites at which DNA unwindin

19、g and initiation of replication occur.lThe replicon model of replication initiationreplicatorinitiatorlInitiation of DNA replicationlin E. colilIn EukaryoteThe replicon model of replication initiation3/18/05 Proposed by Jacob and Brenner in 1963 All the DNA replicated from a particular origin is a r

20、eplicon Two components, replicator and initiator, control the initiation of replicationInitiator protein: specifically recognizes a DNA element in the replicator and activates the initiation of replicationReplicator: the entire site of cis-acting DNA sequences sufficient to direct the initiation of

21、DNA replicationReplicator sequences3/18/05OriC in E. coli chromosomal DNAThree different functions of initiator protein: binds to replicator, distorts/unwinds a region of DNA, interacts with and recruits additional replication factorsDnaA in E. coli (all 3 functions), origin recognition complex (ORC

22、) in eukaryotes (functions 1 & 3)Initiation of DNA replication in E. colilthe sites at which DNA unwinding and initiation of replication occur.lThe replicon model of replication initiationreplicatorinitiatorlInitiation of DNA replicationlin E. colilIn EukaryoteOrigin 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 activation.The regul