Substitution of mucAB or rumAB for umuDC alters the relative frequencies of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and the efficiency of translesion DNA synthesis

Abstract

We have examined the effect of replacing umuDC with mucAB or rumAB on the mutagenic properties of a T-T cyclobutane dimer in an attempt to determine the molecular basis for the differences in UV-induced mutagenesis that are associated with these structurally and functionally related genes. A single-stranded vector carrying a site-specific T-T cis-syn cyclobutane dimer was transfected into a set of isogenic Escherichia coli delta umuDC strains harboring low-copy-number plasmids expressing UmuDC, MucAB, RumAB, or their genetically engineered and mutagenically active counterparts UmuD'C, MucA'B, and RumA'B, respectively. Although the overall mutation frequency was similar for all strains, the relative frequencies of the two classes of mutation induced by the T-T dimer varied according to the mutagenesis operon expressed. In umuDC strains, 3' T-->A mutations outnumbered 3' T-->C mutations, but the reverse was true for the mucAB and rumAB strains. We also found that the T-T dimer was bypassed with differing efficiencies in unirradiated cells expressing wild-type UmuDC, MucAB, and RumAB proteins. These differences can probably be attributed to the relative efficiency of the normal cellular posttranslational activation of UmuD, MucA, and RumA, respectively, since recombinant constructs expressing the mutagenically active UmuD'C, MucA'B, and RumA'B proteins all promoted similarly high levels of bypass in UV-irradiated cells. These results suggest that the UmuD'/UmuC complex and its homologs may differ in their relative abilities to promote elongation from T - T and T - G mismatched termini. Alternatively, they may differentially influence the efficiency with which these mismatches are edited or influence nucleotide insertion by the catalytic subunit of the DNA polymerase III.