ANALYSIS OF RECA MUTANTS WITH ALTERED SOS FUNCTIONS

The Escherichia coli RecA protein has at least three roles in SOS muta genesis: (1) derepression of the SOS regulon by mediating LexA cleavag e; (2) activation of the UmuD mutagenesis protein by mediating its cle avage; and (3) targeting the Umu-like mutagenesis proteins to DNA. Usi ng a combined approach of molecular and physiological assays, it is no w possible to determine which of the three defined steps has been alte red in any recA mutant. In this study, we have focussed on the ability of six particular recA mutants (recA85, recA430, recA432, recA433, re cA435 and recA730) to perform these functions. Phenotypically, recA85 and recA730 were similar in that in lexA(+) and lexA(Def) backgrounds, they exhibited constitutive coprotease activity towards the UmuD muta genesis protein. Somewhat surprisingly, in a lexA(Ind(-)) background, UmuD cleavage was damage inducible, suggesting that the repressed leve l of the RecA protein cannot spontaneously achieve a fully activated state. Although isolated in separate laboratories, the nucleotide sequ ence of the recA85 and recA730 mutants revealed that they were identic al, with both alleles possessing a Glu(38) --> Lys change in the mutan t protein. The recA430, recA433 and recA435 mutants were found to be d efective for both lambda mutagenesis and UmuD cleavage. lambda mutagen esis was fully restored, however, to the recA433 and recA435 strains b y a low copy plasmid expressing the mutagenically active UmuD' protein . In contrast, lambda mutagenesis was only partially restored to a rec A430 strain by a high copy UmuD' plasmid, suggesting that RecA430 may also be additionally defective in targeting the Umu proteins to DNA. S equence analysis of the recA433 and recA435 alleles revealed identical substitutions resulting in Arg(243) --> His. The recA432 mutation had a complex phenotype in that its coprotease activity towards UmuD depe nded upon the lexA background: inducible in lexA(+) strains, inefficie nt in lexA(Ind(-)) cells and constitutive in a lexA(Def) background. T he recA432 mutant was found to carry a Pro(119) --> Ser substitution, a residue believed to be at the RecA subunit interface; thus this comp lex phenotype may result from alterations in the assembly of RecA mult imers.