Reevaluation of the nucleotide cofactor specificity of the RecA protein from Bacillus subtilis.
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.
Steffen
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Department of Biochemistry, School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205, USA.
Bryant
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FR
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases