chapter 25 (part A) DNA metabolism.ppt

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.) 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. 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? …... 4. Autoradiography studies: daughter strands are synthesized immediately after parental strands separate Electron micrographs of the autoradiography of replicating plasmid, SV40 virus, and E. c