Reevaluation of the Nucleotide Cofactor Specificity of the RecA Protein from Bacillus subtilis

Abstract

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...

Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

“…SPP1 on RecA Catalyzed ssDNA-dependent dATP Hydrolysis-dATP hydrolysis was used as an indirect measurement of RecA⅐dATP⅐Mg 2ϩ binding onto SsbA-or heterologous Ssb SPP1 -coated ssDNA. RecA hydrolyzed dATP at a turnover number of ϳ15 min Ϫ1 , which is similar to previously published data on RecA or RecA Eco under similar experimental conditions (5,29). The addition of SsbA to the pre-formed ssDNA⅐RecA⅐dATP⅐Mg 2ϩ complex from subsaturating (1 SsbA/72 nt) to saturating (1 SsbA/18 nt) concentrations partially inhibited (ϳ40%) the dATPase activity of RecA by competing with RecA for ssDNA binding (Fig.…”

“…RecA preferentially hydrolyzes dATP over ATP and supports an efficient DNA strand exchange reaction in the presence of dATP when compared with ATP (5,28,29). Saturating amounts of SsbA, independently of the order of addition, reduce the ssDNA-dependent dATPase activity and block the ATPase activity of RecA (5).…”

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

“…6B, lane 2) (11, 14, 21). However, RecA shows a similar K m for ATP and dATP (21), and the in vivo ATP pool is 100 -500-fold higher than that of dATP both in exponential or post-exponential cells growing under defined medium (Ref. 20 and our unpublished results).…”

“…The ATP pool is 100 -500-fold higher than that of dATP (Refs. 19 and 20 and our unpublished results), but the relative affinity of RecA for ATP or dATP is similar (21), suggesting that the catalytic site of RecA would contain ATP primarily. This premise creates a paradox: RecA⅐ATP can nucleate onto ssDNA, but it cannot catalyze DNA strand exchange (11,12,14).…”

“…RecA hydrolyzes dATP with higher efficiency than ATP when bound to ssDNA (10,11,14,21). To investigate whether changes in the reaction condition can overcome the barrier of the RecA ATPase, the rate of nucleotide hydrolysis was measured at different pH.…”