Characterization of polV_R391: a Y‐family polymerase encoded by rumA′B from the IncJ conjugative transposon, R391

Abstract

SummaryAlthough 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 polV R391, a polV-like Y-family polymerase encoded by rumAЈB from the IncJ conjugative transposon R391. When expressed in a DumuDC lexA(Def) recA730 strain, polVR391 promotes higher levels of spontaneous mutagenesis than the related MucAЈB (polR1) or UmuDЈC (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 3ЈT of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumAЈB, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.

Simple and efficient purification of Escherichia coli DNA polymerase V: Cofactor requirements for optimal activity and processivity in vitro

“…The ability of our various plasmid constructs to promote muta-genesis in vivo was assayed by monitoring spontaneous reversion of the hisG4 (ochre) allele in RW584 [ hisG4, lexA51 ( Def ), recA730 , Δ umuDC ] [28]. Strain RW584 was either transformed with pGB2 (vector) [29], pRW134 ( umuD′C ) [20], pSCD1 ( umuCD′ ), pSCD2 ( his-umuCD′ ), pSCD2-D101N (D101N, umuC104 [30] active site mutant), or pSCD2-DEAA (D101A and E102A, umuC active site substitutions).…”

Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V

“…Similarly, the most error-prone pol V ortholog characterized to date is pol V R391 encoded by rumAB from the integrating conjugating element (ICE) 391 (formerly known as IncJ R391) [62-64]). When located in its native environment (an 88.5kb ICE), pol V R391 promotes very low levels of cellular mutagenesis in E.coli [65].…”

Stress-Induced Mutagenesis

“…However, Pol V homologs with simpler requirements are found on naturally occurring genetic elements, and these polymerases can be used as proxies for Pol V. The IncJ conjugal transposon R391 encodes a Pol V homolog, RumAB, and a more mutagenic form of RumAB, RumA′B, was engineered to model UmuD′C (242). In a strain lacking umuDC , constitutive for the SOS response, and carrying the recA718 allele, which encodes a RecA protein that is constitutively activated (261), overexpression of RumA′B from a low copy plasmid increased the spontaneous mutation frequency in E. coli cells 3- to 12-fold more than expressing UmuD′C from the same plasmid (164). But strains carrying the R394 plasmid, which also encodes a Pol V homolog, have low mutation rates after MMS exposure (128).…”