Intrinsic Polymerase Activities of UmuD′ 2 C and MucA′ 2 B Are Responsible for Their Different Mutagenic Properties during Bypass of a T-T cis-syn Cyclobutane Dimer

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Abstract
In wild-type Escherichia coli , translesion replication is largely dependent upon the UmuD′ 2 C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA′ 2 B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (ɛ subunit) or holE (θ subunit) or carrying the dnaQ allele mutD5 , which is deficient in proofreading but is competent in the structural function of ɛ, or the dnaE antimutator allele spq-2 . The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD′C, MucAB, or MucA′B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA′ 2 B complex is more efficient in promoting translesion replication than the UmuD′ 2 C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA′ 2 B than by UmuD′ 2 C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.