ISOLATION AND CHARACTERIZATION OF NOVEL PLASMID-ENCODED UMUC MUTANTS

Most inducible mutagenesis in Escherichia coli is dependent upon the a ctivity of the UmuDC proteins. The role of UmuC in this process is poo rly understood, possibly because of the limited number of genetically characterized umuC mutants. To better understand the function of the U muC protein in mutagenic DNA repair, we have isolated several novel pl asmid-encoded umuC mutants. A multicopy plasmid that expressed UmuC at physiological levels was constructed and randomly mutagenized in vitr o by exposure to hydroxylamine. Mutated plasmids were introduced into the umu tester strain RW126, and 16 plasmids that were unable to promo te umuC-dependent spontaneous mutator activity were identified by a co lorimetric papillation assay. Interestingly, these plasmid mutants fel l into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasm ids with structural changes in the UmuC protein. Although hydroxylamin e mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mu tations were found in a stretch of 30 amino acids (residues 133 to 162 ) in the middle of the protein, while four other mutations (three of w hich resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutan ts, together with the previously identified chromosomal umuC25, umuC36 , and umuC104 mutations that we have also cloned, should prove extreme ly useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.