BACTERIAL PROTEINS INVOLVED IN DNA REPAIR

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DESCRIPTION (adapted from the investigator's abstract): The primary function of homologous genetic recombination in bacteria is the nonmutagenic repair of arrested replication forks under normal growth conditions. Virtually every replication fork originating at oriC encounters DNA damage at some point, and must undergo recombinational DNA repair. This process represents perhaps the most complex and certainly the least understood of the major pathways for DNA repair. The goal of the work supported by GM52725 is a complete understanding of these critical repair pathways, where the replication and recombination systems are closely integrated. Five specific aims are proposed. First, fundamental biochemistry of several proteins involved in recombinational DNA repair will be explored, focusing on the RecF, RecO, RecR, and RecG proteins. Second, the interaction of a variety of recombination proteins with RecA protein will be explored. This effort will focus on the RecG protein, along with the RuvA and RuvB proteins. In the third aim, DNA substrates will be constructed to mimic at least one of the proposed DNA structures that may occur at arrested replication forks. They will then determine how these DNA structures are processed by recombination enzymes, including the RuvAB complex, the RecG protein, and the RecA proteins. To further elucidate the fate of replication forks when they encounter DNA damage, a fourth aim is to examine the biochemistry of replication fork arrest systematically, using DNA substrate with different types of DNA damage embedded site-specifically. The last aim is to examine the mutagenic replicative bypass of DNA lesions by DNA polymerase V in vitro. These experiments are designed to reconstitute parts of the proposed pathways for replication fork reactivation, to further the ultimate goal of a complete reconstitution with purified enzymes.