The ATP-dependent three-strand exchange activity of the Streptococcus pneumoniae RecA protein (RecA(Sp)), like that of the Escherichia coli RecA protein (RecA(Ec)), is strongly stimulated by the single-stranded DNA-binding protein (SSB) from either E. coli (SSB(Ec)) or S. pneumoniae (SSB(Sp)). The RecA(Sp) protein differs from the RecA(Ec) protein, however, in that its ssDNA-dependent ATP hydrolysis activity is completely inhibited by SSB(Ec) or SSB(Sp) protein, apparently because these proteins displace RecA(Sp) protein from ssDNA. These results indicate that in contrast to the mechanism that has been established for the RecA(Ec) protein, SSB protein does not stimulate the RecA(Sp) protein-promoted strand exchange reaction by facilitating the formation of a presynaptic complex between the RecA(Sp) protein and the ssDNA substrate. In addition to acting presynaptically, however, it has been proposed that SSB(Ec) protein also stimulates the RecA(Ec) protein strand exchange reaction postsynaptically, by binding to the displaced single strand that is generated when the ssDNA substrate invades the homologous linear dsDNA. In the RecA(Sp) protein-promoted reaction, the stimulatory effect of SSB protein may be due entirely to this postsynaptic mechanism. The competing displacement of RecA(Sp) protein from the ssDNA substrate by SSB protein, however, appears to limit the efficiency of the strand exchange reaction (especially at high SSB protein concentrations or when SSB protein is added to the ssDNA before RecA(Sp) protein) relative to that observed under the same conditions with the RecA(Ec) protein.

The RecA protein from the Gram-positive bacterium, Bacillus subtilis, has been reported to catalyze dATP hydrolysis and to promote strand exchange in the presence of dATP but to have no ATP hydrolysis or ATP-dependent strand exchange activity (Lovett, C. M., Jr., and Roberts, J. W. (1985) J. Biol. Chem. 260, 3305-3313). The well characterized RecA protein from Escherichia coli, in contrast, catalyzes the hydrolysis of ATP and dATP at similar rates and can use either ATP or dATP as a cofactor for the strand exchange reaction. To explore this reported difference in nucleotide cofactor specificity in detail, we developed an overexpression system for the B. subtilis RecA protein and purified the protein to greater than 95% homogeneity. Contrary to the previous report, we find that the B. subtilis RecA protein catalyzes the hydrolysis of both dATP and ATP and can perform strand exchange using either dATP or ATP as a cofactor. Our results suggest that the inability of previous investigators to detect the ATP hydrolysis and ATP-dependent strand exchange activities of the B. subtilis RecA protein may have been due to the particular assay conditions that were used in the earlier study.The RecA protein of Escherichia coli (M r 37,842; 352 amino acids) is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. The purified RecA protein will promote a variety of DNA pairing reactions that presumably reflect in vivo recombination functions. The most extensively investigated DNA pairing activity is the ATPdependent three strand exchange reaction in which a circular ssDNA 1 molecule and a homologous linear dsDNA molecule are recombined to yield a nicked circular dsDNA molecule and a linear ssDNA molecule. This reaction proceeds in three phases. In the first phase, the circular ssDNA substrate is coated with RecA protein forming a presynaptic complex that can catalyze the hydrolysis of ATP to ADP and P i ; presynaptic complex formation is stimulated strongly by the E. coli SSB protein, which melts out secondary structure in the ssDNA and allows RecA protein to assemble more efficiently on the ssDNA. In the second phase, the presynaptic complex interacts with a linear dsDNA molecule, and pairing between the circular ssDNA and the complementary strand from the linear dsDNA is initiated.In the third phase, the complementary linear strand is completely transferred to the circular ssDNA by unidirectional branch migration to yield the nicked circular dsDNA and displaced linear ssDNA products (1).The E. coli RecA protein catalyzes the ssDNA-dependent hydrolysis of both ATP and dATP and can use either ATP or dATP as a cofactor for the strand exchange reaction (1-3). Interestingly, the RecA protein from the Gram-positive bacterium, Bacillus subtilis, has been reported to catalyze dATP hydrolysis and to promote strand exchange in the presence of dATP but to have no ATP hydrolysis or ATP-dependent strand exchange activity (4). To study this physiologically and mechanistically intriguing difference...

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