Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli

Abstract

Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example--a general stress-response-controlled switch to error-prone DNA break (double-strand break, DSB) repair--was suggested to be peculiar to an Escherichia coli F' conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F(-) E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response-controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment.

Fig. 1.

Stationary-phase and DSB activation of stress-induced mutation in the E. coli chromosome. (A) Mutation assay. A cleavage site for I-SceI endonuclease (I-site A, red triangle), and tetA allele with a +1-bp frame-shift mutation (tet2, blue arrow) are engineered into the chromosome of cells carrying the chromosomal P_BAD-promoter-regulated I-SceI endonuclease gene (red arrow), which expresses I-SceI slightly (SI Appendix, Fig. S1A) in the absence of glucose (7), the condition used here. Tet^R mutants, caused by compensatory frame-shift mutations (SI Appendix, Fig. S5), occur during starvation in liquid medium without tetracycline (B) and are scored as cfu on rich glucose tetracycline plates after rescue from starvation. oriC and terC, origin and terminus of chromosomal DNA replication. Arrows, 5′ to 3′ orientation of genes. (B) I-SceI–mediated DSBs promote Tet^R reversion in prolonged stationary phase. Strains: CS (cutsite only) (◆); Enz, (enzyme only) (□); DSB (enzyme and cutsite) (■); DSB ΔdinB (×); DSB ΔrpoS (▲). (C) I-SceI–induced DSBs are not mutagenic in unstressed growing cells unless RpoS is up-regulated artificially by deletion of its negative regulator, rssB. The mutagenicity requires RpoS and DinB. In all figures, mutant frequencies are mean ± SEM for at least three independent experiments each, with three cultures per strain per experiment. SI Appendix, Table S5 shows the data in each figure.

Fig. 2.

Proportional contribution of the F′ 128 episome to DSB-induced chromosomal mutation because of the F′ dinB copy. Cultures were assayed for Tet^R mutants after 72 h in stationary phase. Isogenic strains with (red) or without (blue) the F′ 128 plasmid. CS, cutsite only; DSB, enzyme plus cutsite.

Fig. 3.

Genetic requirements of chromosomal DSB-dependent stress-induced mutagenesis in F⁻ cells. (A) DSB-repair/HR proteins, the SOS DNA-damage response, the RpoS general stress response, and DinB/Pol IV are required. Apart from the cutsite-only control (CS), all strains have enzyme and cutsite present (can make DSBs) and are isogenic to the DSB strain except for the mutations indicated. Fold-decreases in mutant frequency, compared with the DSB strain: 58 ± 10 (CS), 25 ± 12 (ΔrpoS), 5.5 ± 0.75 (ΔdinB), 5.2 ± 0.54 (lexAInd⁻, an SOS-induction-defective mutant), 16 ± 3.6 (ΔrecA), 220 ± 130 (ΔrecB), 5.8 ± 0.5 (ruvC), and these genes act in a single pathway (SI Appendix, Fig. S3). (B) DNA Pol II inhibits Pol IV/DinB-dependent mutation, and small Pol V (UmuD′C) requirement for chromosomal Tet^R mutation. The DSB ΔumuCD strain is significantly different from the DSB strain (P = 0.025).

Fig. 4.

Much of spontaneous frame-shift and base-substitution mutation during starvation, in the absence of induced DSBs, is DSB repair-protein DinB- and stress-response–dependent. (A) Frame-shift reversion. The DSB-repair–dependent SIM pathway, requiring RecA, RecB, RuvC, SOS/DinB, and RpoS, constitutes about half of spontaneous Tet^R frame-shift–reversion mutation. Mutant frequencies after 72 h in stationary phase in strains with no I-SceI, and so with only spontaneous DSBs. The mutant strains differ from wild-type significantly: ΔrecA P = 0.001, ΔrecB P = 0.004, ruvC P < 0.001, lexAInd⁻ (SOS⁻) P = 0.001, ΔrpoS P = 0.006, and ΔdinB P = 0.004, Wilcoxon-Mann–Whitney U Test. (B) Half of spontaneous base-substitution mutation (SI Appendix, Fig. S5B) to nalidixic-acid resistance during starvation is DSB-repair protein, DinB- and stress-response–dependent. The mutants differ from wild-type significantly: ΔrecA P < 0.001, ΔrecB P = 0.001, ruvC P < 0.001, lexAInd⁻ (SOS⁻) P = 0.001, ΔrpoS P = 0.01, and ΔdinB P = 0.074.