The Identification of the Single‐Stranded DNA‐Binding Domain of the Escherichia coli RecA Protein

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Abstract
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 sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter‐binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coli RecA in this region did not. The binding of RecA to ssDNA examined using a gel‐shift assay was inhibited by a synthetic peptide derived from this ssDNA‐binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C‐terminus or from N‐terminal residues 6–39. A peptide corresponding to amino acid positions 152– 169 of the RecA molecule and spanning loop LI and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20–amino‐acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region 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 fusion protein containing the putative DNA‐binding site demonstrated significant binding to single‐stranded oligonucleotides in both filter‐binding and gel‐shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA‐binding domain of the RecA protein.