THE IDENTIFICATION OF THE SINGLE-STRANDED DNA-BINDING DOMAIN OF THE ESCHERICHIA-COLI RECA PROTEIN

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from se quences within loops L1 and L2 of the RecA molecule identified from th e crystal structure. Synthetic peptides derived from amino acid residu es 185-219 of several bacterial RecA proteins, which include loop L2 o f RecA, bound to ssDNA in filter-binding assays, whereas three separat e synthetic le point mutants of E. coli RecA in this region did not. T he binding of RecA to ssDNA examined using a gel-shift assay was inhib ited by a synthetic peptide derived from this ssDNA-binding region, bu t not by synthetic peptides derived from amino acid residues 301-329 o f the C-ter minus or from N-terminal residues 6-39, A peptide correspo nding to amino acid positions 152-169 of the RecA molecule and spannin g loop L1 and its flanking regions did not bind ssDNA at peptide conce ntrations up to 250 mu M. We have also defined a synthetic 20-amino-ac id peptide that comprises amino acid residues 193-212 and includes loo p L2 of RecA as the minimum unit that can bind to ssDNA from this regi on of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185-224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303 -353). In contrast to the C-terminus-derived fusion protein, the fusio n protein containing the putative DNA-binding site demonstrated signif icant binding to single-stranded oligonucleotides in both filter-bindi ng and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193-212 is either parr of or the whole ssDNA-binding domain of the RecA protein.