Escherichia coli cells expressing a mutant glyV (glycine tRNA) gene have a UVM-constitutive phenotype: implications for mechanisms underlying the mutA or mutC mutator effect

Abstract

Transfection of M13 single-stranded viral DNA bearing a 3,N 4-ethenocytosine lesion into Escherichia coli cells pretreated with UV results in a significant elevation of mutagenesis at the lesion site compared to that observed in untreated cells. This response, termed UVM, for UV modulation of mutagenesis, is induced by a variety of DNA-damaging agents and is distinct from known cellular responses to DNA damage, including the SOS response. This report describes our observation, as a part of our investigation of the UVM phenomenon, that E. coli cells bearing a mutA or mutC allele display a UVM-constitutive phenotype. These mutator alleles were recently mapped (M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380-4385, 1996) to the glyV (mutA) and glyW (mutC) tRNA genes. Each mutant allele was shown to arise by an identical mutation in the anticodon sequence such that the mutant tRNAs could, in principle, mistranslate aspartate codons in mRNA as glycine at a low level. Because a UVM-constitutive phenotype resulting from a mutation in a tRNA gene was unexpected, we undertook a series of experiments designed to test whether the phenotype was indeed mediated by the expression of mutant glycine tRNAs. We placed either a wild-type or a mutant glyV gene under the control of a heterologous inducible promoter on a plasmid vector. E. coli cells expressing the mutant glyV gene displayed all three of the following phenotypes: (i) missense suppression of a test allele, (ii) a mutator phenotype measured by mutation to rifampin resistance, and (iii) a UVM-constitutive phenotype. These phenotypes were not associated with cells expressing the wild-type glyV gene or with cells in which the mutant allele was present but was not transcriptionally induced. These observations provide strong support for the idea that expression of mutant tRNA can confer a mutator phenotype, including the UVMconstitutive phenotype observed in mutA and mutC cells. However, our data imply that low-level mistranslation of the epsilon subunit of polymerase III probably does not account for the observed UVM-constitutive phenotype. Our results also indicate that mutA ⌬recA double mutants display a normal UVM phenotype, suggesting that the mutA effect is recA dependent. The observations reported here raise a number of intriguing questions and raise the possibility that the UVM response is mediated through transient alteration of the replication environment.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance

“…One possible explanation for this could be mistranslationinduced error-prone replication leading to an increased mutation rate, a phenomenon described in Escherichia coli (18,19). To test whether this was the case, we sequenced the RRDR of rpoB and found that 24/24 of the EMED colonies isolated from media containing rifampicin were WT in sequence.…”

Requirement for Homologous Recombination Functions for Expression of the mutA Mistranslator tRNA-Induced Mutator Phenotype in Escherichia coli

“…In addition to the well-known Escherichia coli SOS system, emerging evidence suggests the existence of a number of such pathways in E. coli (7). One of the more intriguing newly recognized mutagenic pathways is the one elicited in mutA cells (7,16,27), in which the expression of an altered glyV glycine tRNA gene results in a strong mutator phenotype (27) characterized by elevation of transversions. In the mutA allele, the normal 3Ј-CCG anticodon is mutated to a 3Ј-CUG anticodon such that the mutant tRNA misreads the aspartate codon 5Ј-GAU/C as glycine at a low efficiency.…”

“…Expression of the mutA phenotype is constitutive and requires the recA and recB genes, but not umuD, umuC, dinB, or other lexA-repressible functions (16,23), and thus represents a novel inducible mutagenic pathway termed "translational stress-induced mutagenesis" (TSM) (7).…”

“…2B for chemical structure) borne on M13 single-stranded DNA (ssDNA) vectors transfected into E. coli cells (16,23). In this assay, M13 ssDNA bearing a single sitespecific lesion (εC-ssDNA) is transfected into an appropriate strain, and the resulting progeny phage are analyzed for mutations at the εC site by a quantitative multiplex sequence analysis procedure summarized in Fig.…”

“…In this assay, M13 ssDNA bearing a single sitespecific lesion (εC-ssDNA) is transfected into an appropriate strain, and the resulting progeny phage are analyzed for mutations at the εC site by a quantitative multiplex sequence analysis procedure summarized in Fig. 2C (16,17,19,21). The assay depends on limited elongation of a prelabeled primer to characteristic lengths, depending on the base replacing the lesion upon replication.…”