SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions

Abstract

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N4-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. colistrains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA‐dependent error‐prone DNA replication activity

“…We have demonstrated recently that SOS induction can independently elevate mutagenesis at ɛC, but with a mutational specificity different from that observed in mutA cells. Thus, in fully SOS‐induced cells, C → T mutations are equal to or exceed C → A mutations, whereas in mutA cells (and UVM‐induced cells), C → A mutations predominate (Rahman and Humayun, 1999). The specificity seen for mutA polA cells is consistent with a partial induction of SOS superimposed on the mutA mutator phenotype.…”

The Escherichia coli UVM response is accompanied by an SOS‐independent error‐prone DNA replication activity demonstrable in vitro

“…Almost all the enhanced mutagenesis at εC residues observed in UVM‐induced cells is accounted for by an increase in C → A and C → T mutations, with C → A mutations predominating over C → T mutations. By uncoupling SOS mutagenesis from DNA damage, we have demonstrated recently that mutagenesis at εC can be elevated independently by the SOS pathway (Rahman and Humayun, 1999). Although the magnitude of mutagenesis induced by the SOS and UVM pathways is similar, there is an interesting difference in specificity.…”

“…Both C → A and C → T mutations are represented, with C → A mutations predominating in uninduced as well as induced cell extracts. These results compare with the following in vivo data: mutation fixation at εC in uninduced cells ranges from 5% to 10% and is elevated in induced cells to about 50% (Palejwala et al ., 1994; Wang et al ., 1995; Rahman and Humayun, 1999). Thus, the overall patterns are similar, but not identical, in vitro and in vivo .…”

“…The existence of the UVM‐like mutagenic phenomena, as well as of the NSD genes, is consistent with the idea that E. coli does not rely on a single pathway for the transient expression of mutator activities, but rather is capable of deploying multiple such pathways to fine tune its response to environmental or physiological conditions. For example, even though the SOS pathway is absolutely required for the bypass of abasic sites (a common form of DNA damage), mutagenesis at abasic sites is elevated further by the UVM pathway (Rahman and Humayun, 1999). On the other hand, mutagenesis at εC lesions can be elevated independently by no less than three inducible pathways, namely, SOS, UVM and TSM, of which the first two are DNA damage inducible, whereas the third (translational stress‐induced mutagenesis) may be induced by mistranslation (Humayun, 1998).…”

“…The presence of the Δ( umuDC ) 595 allele in AM122 was confirmed by PCR amplification (forward primer 5′‐TGGCGAATGCTTC TATTCTATT; reverse primer 5′‐CGGTATTTTTATCGCT TACCTG) followed by an analysis of restriction digestion patterns of the PCR products. Strain AM127 was characterized by UV sensitivity (at 30 J m −2 ) conferred by the Δ( umuDC ) 595 allele (Rahman and Humayun, 1999). E. coli strain AM123 was created by transferring the Δ dinB ::kan r allele from strain YG7207 to strain AM122 by P1 transduction and selection on an LB plate containing 80 µg ml −1 kanamycin (Sigma).…”