Bacterial Proteins Involved in DNA Repair

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DESCRIPTION (provided by applicant): The primary function of homologous genetic recombination in bacteria is the nonmutagenic repair of arrested replication forks. Virtually every replication fork originating at oriC encounters DMA 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 work supported by GM52725 has three goals, all directed at a complete understanding of replication fork repair pathways, where the replication and recombination systems are closely integrated. The work is currently focused on the processes that regulate the function of the RecA protein. The first specific aim constitutes the major portion of the effort. The fundamental biochemistry of proteins involved in the regulation of RecA protein will be explored, focusing on the RecFOR, RecX, Dinl, RdgC, PsiB, UvrD, and RecQ proteins. RecA is regulated on multiple levels, and this effort is designed to systematically explore that regulation. An interwoven system of regulatory activities modulating almost every aspect of RecA function has been outlined in recent studies. The other two aims represent a smaller investment in effort, but allow for an integration and expansion of the information obtained in aim 1. The second aim is to reconstitute a key step in some fork repair pathways, called fork regression. This effort relies on the construction of novel DNA substrates that mimic the structure of stalled forks. The effort will draw heavily on the information provided under aim 1. The final aim is to examine the mutagenic replicative bypass of DNA lesions by DNA polymerase V in vitro. Here we are redefining the role of RecA in this repair process and potentially exploring a novel aspect of RecA regulation. These experiments will provide new information about critical parts of the pathways leading to replication fork reactivation. The ultimate goal is a complete reconstitution of the pathways with pure enzymes.