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 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 L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 microM. 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.
Gardner
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RV
Genetics and Biochemistry Branch, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
Voloshin
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ON
Camerini-Otero
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RD
D000595:Amino Acid Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism*; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D011938:Rec A Recombinases