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.
Highlights
The RecA protein of Escherichia coli (Mr 37,842; 352 amino acids) is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA
The circular ssDNA substrate is coated with RecA protein forming a presynaptic complex that can catalyze the hydrolysis of ATP to ADP and Pi; 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
Our results demonstrate that the RecA protein from B. subtilis can catalyze the ssDNA-dependent hydrolysis of both dATP and ATP
Summary
Materials—E. coli RecA protein was prepared as described previously (5). E. coli SSB was provided by Dr Roger McMacken (Johns Hopkins University). The pellet was suspended in R buffer, 0.5 M NaCl and dialyzed overnight against R buffer, 0.5 M NaCl. The dialysate was centrifuged at 27,000 ϫ g for 15 min, and the supernatant was loaded onto a HiTrap Blue column (5 ml, Amersham Pharmacia Biotech) that had been equilibrated with R buffer, 0.5 M NaCl. The column was washed with 0.5 M NaCl (40 ml), 1.0 M NaCl (40 ml), and 2.0 M NaCl (80 ml). The dialysate was loaded onto a HiTrap Heparin column (10 ml, Amersham Pharmacia Biotech) and eluted with a 120-ml linear gradient of R buffer, 0.1–1.0 M NaCl. The protein-containing fractions (centered at 0.28 M NaCl) were pooled and dialyzed overnight against R buffer, 20% glycerol, 0.1 M NaCl to yield the final fraction (13 mg) of purified, nuclease-free B. subtilis RecA protein (Fig. 1). Amino-terminal protein sequencing was carried out by the Johns Hopkins Protein/ Peptide Sequencing Facility
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