Direct analysis of fork blockage and DNA repair in vivo

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DESCRIPTION: (provided by applicant): A number of labs have advanced our understanding of how DNA damage is processed by constructing synthetic DNA molecules that contain site-specific/chemically specific damage and transforming these molecules into cells. The ability of the damaged molecules to generate viable progeny or particular mutant progeny is then analyzed. While these approaches have provided much useful knowledge, they are limited because the progeny DNA is analyzed only after many rounds of propagation. The experiments described in this proposal are designed to allow the direct physical analysis of DNA containing site-specific damage upon introduction into living bacterial cells. The approach utilizes a specific mutant of phage X that makes small heads, which package only about 15 kb of DNA. This is a size small enough to permit construction of molecules containing site-specific damage. Damaged DNA, containing radioactive residues for ease of analysis, will be packaged in vitro into the small heads, and assembled into phage particles. The particles will be purified and then used to infect cells. DNA will be prepared from the infected cells as a function of time to follow the fate of the damage. Repair of the damage will be followed kinetically using restoration of a restriction enzyme cleavage site at the location of the damage. In addition, a major interest of my lab is to determine whether replication forks are blocked at the damage using 2-dimensional gel electrophoresis. The experiments described here will attempt to validate this system using DNA with and without abasic sites, which are very important in both spontaneous and induced DNA damage. We hope to be able to measure the kinetics of abasic site repair in wild type and mutant strains, and also to determine whether abasic sites block replication forks. If the system is validated, many other kinds of DNA damage can presumably be analyzed in-future studies.