Our specific aims seek to extend our knowledge about the role of nucleases in recombination, repair and mutation avoidance. Maintenance of genomic stability is important to all organisms and defects in these processes lead to cancer-predisposition in several defined human syndromes. Many of the mechanisms that ensure genetic stability are conserved in prokaryotes and eukaryotes. Our studies will employ both biochemical and genetic approaches using the bacterium E. coli. 1. RecJ is a 5' ssDNA exonuclease involved in DNA recombination, repair and mutation avoidance. It is the best-characterized member of a large group of proteins with a novel structure.
Our first aim will be to examine some questions raised by the crystal structure of RecJ, namely, the nature of the metal binding site, its interaction with DNA and the structural basis of processivity. 2.
Our second aim seeks elucidate the mechanism of template-switch mutations in quasipalindromes and illegitimate recombination reactions at very short homologies. We have shown that both processes are normally circumvented by single-strand DNA exonucleases. Both types of processes may contribute significantly to mutagenesis and genomic evolution. We will develop assays to examine cis-and trans-acting factors on these mutagenic processes. Other experiments to find natural vulnerable sites for such mutations will help establish how these processes contribute to mutational burden of E. coli. 3.
Our third aim i s to pursue the genetic and biochemical function of the bacterial RadC protein. This protein shares a duplicated helix-hairpin-helix motif characteristic of proteins that bind distorted DNA, particularly the Radl/XPF/Mus81 class of proteins that cleave branched structures in DNA. We will examine RadC's activity on a variety of branched structures and its potential functional and physical interaction with other proteins. We will extend genetic analysis of radC to determine what role it may play in repair and recombination.
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