The goals of this research proposal are to elucidate and characterize fundamental interactions that are important for the activities of the RecA protein of Escherichia coli. This protein is required for homology dependent recombination, for regulating a set of genes that participate in a DNA damage stress response (SOS response) and for participating directly in the biochemical process leading to mutagenesis. The basic interactions that will be investigated are those involved in i) RecA protein subunit recognition, ii) ATP binding and hydrolysis, and iii) recognition and binding to other proteins such as lambdoid repressors, the cellular LexA repressor and the umuD protein which is needed for a major pathway of chemical mutagenesis. The sequence(s) within the first 90 residues of RecA protein required for subunit binding will be investigated using affinity chromatography and fusion proteins between RecA and beta-lactamase. Binding of synthetic peptides corresponding to the regions of interest will be studied. Two substitution mutations (Tyr264-Phe264 and Tyr264-Ser264) in the ATP binding site have been constructed by site-directed mutagenesis and will be fully characterized with respect to ATP hydrolysis, DNA binding and the ability to carry out DNA pairing (annealing, strand transfer). Second site suppressor mutations will be isolated and characterized in order to identify other important elements of the ATP binding domain. In addition, site-directed mutagenesis will be used to alter residues with a conserved sequence in TecA protein that is found in a number of nucleotide binding proteins. The properties of the proteins will be characterized in vitro. The interaction of RecA protein-polynucleotide complexes with other cellular proteins will be investigated, in particular, the role of Gly204 in LexA protein binding will be examined. Finally, the role of RecA-like activity in homologous recombination and DNA repair will be investigated by constructing yeast strains lacking the gene encoding this function. These latter experiments will provide additional insight into the mechanisms of recombination in eurkaryotic cells.
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