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  • Title: Analysis of recA mutants with altered SOS functions.
    Author: Ennis DG, Levine AS, Koch WH, Woodgate R.
    Journal: Mutat Res; 1995 Jan; 336(1):39-48. PubMed ID: 7528894.
    Abstract:
    The Escherichia coli RecA protein has at least three roles in SOS mutagenesis: (1) derepression of the SOS regulon by mediating LexA cleavage; (2) activation of the UmuD mutagenesis protein by mediating its cleavage; and (3) targeting the Umu-like mutagenesis proteins to DNA. Using a combined approach of molecular and physiological assays, it is now possible to determine which of the three defined steps has been altered in any recA mutant. In this study, we have focused on the ability of six particular recA mutants (recA85, recA430, recA432, recA433, recA435 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 mutagenesis protein. Somewhat surprisingly, in a lexA(Ind-) background, UmuD cleavage was damage inducible, suggesting that the repressed level of the RecA* protein cannot spontaneously achieve a fully activated state. Although isolated in separate laboratories, the nucleotide sequence of the recA85 and recA730 mutants revealed that they were identical, with both alleles possessing a Glu38-->Lys change in the mutant protein. The recA430, recA433 and recA435 mutants were found to be defective for both lambda mutagenesis and UmuD cleavage. lambda mutagenesis was fully restored, however, to the recA433 and recA435 strains by a low copy plasmid expressing the mutagenically active UmuD' protein. In contrast, lambda mutagenesis was only partially restored to a recA430 strain by a high copy UmuD' plasmid, suggesting that RecA430 may also be additionally defective in targeting the Umu proteins to DNA. Sequence analysis of the recA433 and recA435 alleles revealed identical substitutions resulting in Arg243-->His. The recA432 mutation had a complex phenotype in that its coprotease activity towards UmuD depended upon the lexA background: inducible in lexA+ strains, inefficient in lexA(Ind-) cells and constitutive in a lexA(Def) background. The recA432 mutant was found to carry a Pro119-->Ser substitution, a residue believed to be at the RecA subunit interface; thus this complex phenotype may result from alterations in the assembly of RecA multimers.
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