On the in vivo function of the RecA ATPase.
The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.
Campbell
M J
MJ
Department of Biochemistry Beckman Center, Stanford Medical Center, 279 Campus Drive, Palo Alto, CA, 94304-5307, USA. campbell@mag.com
Davis
R W
RW
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000502:physiology*; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D000099160:LexA Repressor Protein