Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity.
The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue. The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein. Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2. To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue. The new [Asn160]recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein. This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain. Although it is an active single-stranded DNA-dependent ATPase, the [Asn160]recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro. Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the [Asn160]recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E. coli protein. To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the [Asn160]recA protein.
Bryant
F R
FR
Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D005998:Glycine; D007700:Kinetics; D009154:Mutation; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D014466:Ultraviolet Rays
D001224:Aspartic Acid; D004254:DNA Nucleotidyltransferases; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; C037693:single stranded DNA dependent ATPase; D005998:Glycine