ERROR PRONE REPAIR--MUTAGENESIS AND AGING

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The proposed project represents the first step towards our long term goal, which is the understanding, on a molecular level, of the role of mutagenesis mechanisms in aging. Since mutagenesis mechanisms in mammalian systems are largely obscure, and even in prokaryotes the mechanisms of some major mutagenesis pathways are unknown, we propose to begin by studying the molecular mechanism of SOS-mutagenesis (also termed error-prone repair), and in particular UV-mutagenesis in E. coli. The unique interest in this process stems from the surprising finding that it is an active mutagenesis process which requires the induction of specific gene product i.e., recA, umuC, umuD and maybe more. Our strategy, adopting primarily a biochemical approach, is to utilize complete in vitro replication systems for the replication of UV-irradiated DNA, using both wild-type and SOS-induced cells as the proteins sources, and assay for nascent DNA length or the production of base changes. We also propose to develop a methodology for the preparation of special DNA substrates which will contain a single pyrimidine photodimer (the major UV lesion) at a specific site. These will be used to study directly the role of the major UV-lesion in mutagenesis in vivo and in vitro. Purified replication systems, developed primarily by A. Kornberg and his colleagues will be used throughout the study. These will include replication of single stranded DNA from phages M13 and G4 to the replicative form (SS leads to RF reaction) and replication of replicative form I DNA of phage Phi-X174 to single stranded DNA (FR leads to SS reaction). A detailed biochemical analysis of bypass of, and termination at pyrimidine photodimers by DNA polymerase III holoenzyme, the major replicative polymerase of E. coli, will be carried out. These studies are to be followed by an attempt to elucidate the role of S.O.S. induced proteins such as umuC and umuD in UV-mutagenesis. Understanding error-prone repair in E. coli will give us clues and some starting guidelines for investigating similar processes in mammalian systems, and the methodologies developed will be applied for the actual study of mutagenesis in mammlians, and its role in aging.