Characterization of polV: a Y-family polymerase encoded by rumA′B from the IncJ conjugative transposon, R391R391: a Y-family polymerase encoded by rumA′B from the IncJ conjugative transposon, R391

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Abstract

Summary

Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polVR391, a polV-like Y-family polymerase encoded by rumA′B from the IncJ conjugative transposon R391. When expressed in a ΔumuDC lexA(Def) recA730 strain, polVR391 promotes higher levels of spontaneous mutagenesis than the related MucA′B (polR1) or UmuD′C (polV) polymerases respectively. Analysis of the spectrum of polVR391-dependent mutations in rpoB revealed a unique genetic fingerprint that is typified by an increase in C:G→A:T and A:T→T:A transversions at certain mutagenic hot spots. Biochemical characterization of polVR391 highlights the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polVR391 can also bypass a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3′T of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumA′B, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.

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