《生物化学》课程PPT教学课件（英文版）chapter 25 DNA metabolism

Chapter 25 DNA Metabolism 1. How does a DNA molecule replicate with high fidelity? 2. How are DNA lesions(damages) repaired to maintain the integrity of genetic information? 3. How do dNA molecules recombine rearrange )? High accuracy, multitude of participants

Chapter 25 DNA Metabolism 1. How does a DNA molecule replicate with high fidelity? 2. How are DNA lesions (damages) repaired to maintain the integrity of genetic information? 3. How do DNA molecules recombine (rearrange)? High accuracy, multitude of participants

1. The deduced double helix structure of DNA revealed the possible ways for its replication(1953) Each DNA strand was proposed to act as the template(complement) of the other The way a DNA molecule replicates was hypothesized to be semiconservative: each of the newly synthesized DNA duplexes consists of one strand from the parent DNa and one strand of newly synthesized Watson and Crick, 1953).(the conservative replication would generate two daughter DNA molecules with one consisting of two new and one of two old strands

1. The deduced double helix structure of DNA revealed the possible ways for its replication (1953) • Each DNA strand was proposed to act as the template (complement) of the other. • The way a DNA molecule replicates was hypothesized to be semiconservative: each of the newly synthesized DNA duplexes consists of one strand from the parent DNA and one strand of newly synthesized (Watson and Crick, 1953). (the conservative replicationwould generate two daughter DNA molecules with one consisting of two new and one of two old strands.)

The hypothesis of semiconservative replication proposed by Watson and Crick in1953 GG New strandI Old strand 于TA Old New New Old

New strand Old strand The hypothesis of semiconservative replication proposed by Watson and Crick in 1953

2. DNA replication was proved to be semiconservative by the Meselson-Stahl experiment using E coli cells (1957) 5N (the Heavy isotope) and 4N (the Light isotope) was used (as NH4CI)to label the DNa to distinguish the old and newly synthesized DNa molecules in cells Three types of DNa molecules containing various proportions of 5N and 4N(H-H, H-L, L-L)were separated by centrifugation to equilibrium in a cesium chloride(CscI) density gradient(only two types would be expected if replication is conservative)

2. DNA replication was proved to be semiconservative by the Meselson-Stahl experiment using E. coli cells (1957) • 15N (the Heavy isotope) and 14N (the Light isotope) was used (as NH4Cl) to label the DNA to distinguish the old and newly synthesized DNA molecules in cells; • Three types of DNA molecules containing various proportions of 15N and 14N (H-H, H-L, L-L) were separated by centrifugation to equilibrium in a cesium chloride (CsCl) density gradient (only two types would be expected if replication is conservative)

DNA extracted and centrifu to equilibrium in CsCl R density gradient radioisotope labeling and density gradient centrifugation clearly (a) He eavy DNA (ON)- distinguishes Original parent molecule replications of semiconservative Hybrid DNA from conservative (b)(15N14N) daughter molecules Light DNA(N)- (e)Hybrid dNA daughter molecules

Radioisotope labeling and density gradient centrifugation clearly distinguishes replications of semiconservative from conservative

N-15N Bottom I0 generation The meselson Stahl experiment: DNA molecules AI generation duplicate semiconservative in E coli cells 白2 generations 白3 generations A 4 generations - 0 and 2 mixed e0 and 4 mixed 14N-14N15N-14N15N-5N

The Meselson￾Stahl experiment: DNA molecules duplicate semiconservatively in E. coli cells. 15N- 15N 0 generation 1 generation 2 generations 3 generations 4 generations 0 and 2 mixed 0 and 4 mixed 15N- 14N 15N- 14N- 14N 15N Top Bottom

3. A variety of simple questions were asked about DNA replication Are the two parental strands completely unwound before replication begins Does replication begin at random sites or at unique sites Does DNA replication proceed in one direction or both directions? The overall chain growth occurs in 5-3,35,or both directions? What mechanisms ensure that DNA replicates once per cell division?

3. A variety of simple questions were asked about DNA replication • Are the two parental strands completely unwound before replication begins? • Does replication begin at random sites or at unique sites? • Does DNA replication proceed in one direction or both directions? • The overall chain growth occurs in 5` 3`, 3` 5`, or both directions? • What mechanisms ensure that DNA replicates once per cell division?

What enzymes take part in DNA synthesis? How does duplication of the long helical duplex occur without the strands becoming tangled?

• What enzymes take part in DNA synthesis? • How does duplication of the long helical duplex occur without the strands becoming tangled? …

4. Autoradiography studies: daughter strands are synthesized immediately after parental strands separate Electron micrographs of the autoradiographs of replicating plasmid, SV40 virus, and E coli chromosomal DNA with 3H-thymidine incorporated revealed @-like structures: no single stranded DNa was visible The chromosomal DNA of coli is a single huge circle! No temporary creation of linear DNA occurred during replication of the circular dNa

4. Autoradiography studies: daughter strands are synthesized immediately after parental strands separate • Electron micrographs of the autoradiographs of replicating plasmid, SV40 virus, and E. coli chromosomal DNA with 3H-thymidine incorporated revealed -like structures: no single stranded DNA was visible. • The chromosomal DNA of E. coli is a single huge circle! • No temporary creation of linear DNA occurred during replication of the circular DNA

A electron micrograph of the replication intermediate of a plasmid DNA: 6-shaped structures were observed: no single stranded dNA is visible No complete unwinding of the two parental strands occurred before the daughter strands are synthesized

A electron micrograph of the replication intermediate of a plasmid DNA: -shaped structures were observed; no single stranded DNA is visible. No complete unwinding of the two parental strands occurred before the daughter strands are synthesized