Our goals are to elucidate the molecular features of the structure, of the E. coli recA protein and relate these structural details to the enzymatic properties of the protein. The approaches to be taken include a combination of biochemical modification studies, recombinant DNA techniques and 'classical' microbial genetics. Biochemical investigations of recA protein include the direct photolinking of DNA to the polynucleotide binding site of recA protein. Identification of the regions of recA protein covalently crosslinked to DNA will be accomplished using tryptic digestion, HPLC and amino acid analyses. Using a similar approach the ATP binding site of recA protein has been identified and we now wish to identify the amino acid residue(s) to which ATP is covalently linked. This will be accomplished by further proteolytic digestion of the modified peptide and amino acid sequence analysis. Using photoaffinity labeling and tryptic mapping techniques we will investigate the conservation of this ATP binding domain among five heterologous recA proteins derived from P. vulgaris, E. carotovora, S. typhimurium, S. flexneri and E. coli B/r. The genes for these heterologous proteins have been cloned into E. coli K12 and are expressed at high levels in appropriate genetic backgrounds. This comparative analysis will provide valuable information on the nature of recA protein ATP interaction. The information gained by biochemical modification studies of domains of recA protein will permit a more detailed functional analysis through the application of site-directed mutagenesis techniques to the appropriate region of the recA structural gene. Both Ba131 deletion and oligonucleotide-directed mutagenesis will be employed to modify those regions of recA protein that are important for substrate binding. We will then analyze the effects of these modifications or the functions of recA protein both in vitro and in vivo. Additional information on the domains of recA will be obtained by DNA sequence analysis of the cloned recA genes from P. vulgaris, E. carotovora, S. typhimurium, S. flexneri and E. coli B/r. The ability of the cloned genes to complement defects of an E. coli K12 recA- mutation indicates conservation of important functional domains among these heterologous proteins.
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