Mismatch repair protein MutL becomes limiting during stationary-phase mutation

Abstract

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.

Figure 1

Overproduction of MutL alone, or with MutS, depresses stationary-phase Lac⁺ reversion. Plasmids [pControl], [pMutL&MutS], [pMutS], and [pMutL] are pSL4, pSL7, pSL6, and pSL5, respectively (Materials and Methods). Error bars represent one standard error of the mean (s.e.m.) and are smaller than the data point where not visible.

Figure 2

Inhibition of stationary-phase Lac⁺ reversion in a hypermutable recD strain. Plasmids [pControl], and [pMutL&MutS] are pSL4, and pSL7, respectively (Materials and Methods). Error bars as in Fig. 1.

Figure 3

Representative quantitative Western immunoblots. Data from multiple experiments of this type are summarized and described in Fig. 4. (A) MutL levels. (Lanes 1–4) Quantification standards (Feng et al. 1996; see Materials and Methods; legend to Fig. 4): 0, 4.5, 9, and 18 ng of His₆–MutL protein, respectively; (lanes 5–10) MutL in cultures grown exponentially (9.9 ng) and at days 0 (12.9 ng), 2 (12.5 ng), 4 (12.1 ng), 6 (9.6 ng), and 8 (9.9 ng), respectively. (B) MutS levels. (Lanes 1–5) Quantification standards: 0, 2.5, 5, 10, and 20 ng of His₆–MutS protein, respectively; (lanes 6–11) MutS in cultures grown exponentially (15.4 ng) and at days 0 (10.8 ng), 2 (7.7 ng), 4 (6.2 ng), 6 (4.8 ng), and 8 (4.3 ng), respectively. (C) MutH levels. (Lanes 1–4) Quantification standards: 0, 0.25, 0.5, and 1.0 ng of His₆–MutH protein, respectively; (lanes 5–10) MutH in cultures grown exponentially (0.78 ng) and at days 0 (0.54 ng), 2 (0.52 ng), 4 (0.44 ng), 6 (0.47 ng), and 8 (0.36 ng), respectively.

Figure 4

Amounts of MutL, MutS, and MutH proteins in growing, stationary-phase, and starved cells carrying MutL- and MutS-overproducing plasmids. Data are summaries of quantifications from Western blots performed as in Feng et al. (1996; see Materials and Methods). Days 0–8 indicate days after plating the stationary-phase lac⁻ cells on lactose medium (Materials and Methods). (E) Exponential cultures. At least three experiments were performed. Each histogram bar represents the mean (error bars, s.e.m.). (Solid bars) pControl; (open bars) pMutL; (hatched bars) pMutS; (shaded bars) pMutL and pMutS. (A) MutL levels. (B) MutS levels. For MutH, two different sets of experiments are shown. (C) The first was measured in nanograms of MutH protein per 150 μg of total cellular protein. (D) The second set was measured as numbers of MutH monomers per cell [see Materials and Methods for the recalibration of the number of MutH monomers per cell with respect to previous results (Feng et al. 1996). This is discussed further in the text.