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J Bacteriol. 2000 Sep; 182(17): 5025–5028.
PMCID: PMC111390

Reduction of GC → TA Transversion Mutation by Overexpression of MutS in Escherichia coli K-12


Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC → TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC → TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT → CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.

The MutS homodimer binds to mismatched bases, small bulge loops, and damaged bases as an initial step in methyl-directed DNA mismatch repair in Escherichia coli (2, 4, 7, 22, 26, 27, 29, 30, 34). MutS is also thought to play roles in methyl-independent repair pathways, such as very-short-patch repair (18). The amount of MutS is strongly down regulated as E. coli cells enter stationary phase (9, 31). This regulation is partly mediated at the level of mRNA stability by the RNA chaperone protein Hfq, which acts to destabilize the mutS transcript (31). Down regulation of the amount of MutS in stationary-phase cells may help to coordinate repair capacity with decreased DNA replication (9, 31). In addition, decreased amounts of MutS could contribute to increased mutagenesis or homeologous recombination in stationary-phase cells or in bacterial cells that resume growth after stationary phase. Transfection experiments of phage DNA containing heteroduplexes suggest that mismatch repair capacity is indeed decreased in cultures grown overnight and that this reduced repair capacity can be reversed by overexpression of MutS but not MutL (5).

To investigate further possible effects of the amount of MutS on mutation rate, we determined mutation rates in E. coli cells that gradually enter stationary phase. Hall devised a papillation assay to determine the rates of mutation as the Cupples-Miller base substitution and frameshift tester strains enter and remain in stationary phase (8, 12). In this assay, bacteria are spread onto plates containing 0.01% glycerol, 0.1% lactose, and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) at 30°C. Bacterial colonies stop growing when they exhaust the limited glycerol; however, blue lacZ+ papillae continue to appear gradually within colonies. In contrast to the abrupt cessation of growth after carbon source deprivation, which is used routinely to determine adaptive mutagenesis (10, 15), the total viable bacteria per day increased very slowly in this papillation assay (data not shown). Hall reported that of the possible transitions, transversions, and frameshifts, the most frequent mutation in stationary-phase bacteria was the GC → TA transversion in tester strain CC104 (12). We obtained results similar to those of Hall for CC104 and CC104 containing an empty cloning vector (pVector) (Fig. (Fig.1)1) (15).

FIG. 1
Overexpression of MutS and to a lesser extent MutL decreased the number of lacZ GC → TA transversion mutants in E. coli strain CC104 in stationary-phase colonies. Experiments were done essentially by the method of Hall (12). CC104 containing the ...

Unexpectedly, we found that overexpression of MutS, and to a lesser extent MutL, significantly decreased GC → TA transversion in CC104 under these experimental conditions (Fig. (Fig.1)1) (15). MutL is an ATPase that interacts with DNA, MutS, MutH, and UvrD during methyl-directed mismatch repair (3, 11, 13, 14, 28, 34, 35). Previously, we demonstrated that pMutS and pMutL overexpress MutS and MutL by about 60- and 20-fold, respectively, in growing and starved bacteria (15). In contrast to CC104, MutS or MutL overexpression did not significantly affect the appearance of lacZ+ papillae by frameshift tester CC107 (data not shown), which is extremely sensitive to defects in methyl-directed mismatch repair (8). Transition mutation testers CC102 (GC → AT) and CC106 (AT → GC), which are also sensitive to defects in methyl-directed mismatch repair (8), were not used in these assays. In our hands, the background color of CC102 colonies was too blue to detect papillae reliably (data not shown), and the number of lacZ+ papillae formed by CC106 colonies was very low, even after prolonged incubation (12).

Control experiments indicated that CC104(pVector), CC104(pMutS), and CC104(pMutL) formed the same number of total viable cells per plate per day to 4 days of incubation after which lacZ+ papillae formation became significant for CC104(pVector) (Fig. (Fig.1;1; data not shown). We used CC101, which is the tester for AT → CG transversion (8), to determine the total number of viable cells per plate to 8 days, because CC101, CC101(pVector), CC101(pMutS), and CC101(pMutL) are isogenic, except for the lacZ allele, grew at rates similar to those of the corresponding CC104 derivatives, and formed a negligible number of lacZ+ papillae in this assay (data not shown) (12). We did not use a P90C [F′ Δ(lacI lacZ)] strain, which should have been isogenic to CC101 and CC104, because our isolate of this strain exhibited different growth characteristics from those of CC101 and CC104 on these media (data not shown). The number of total viable cells per day was used to convert data such as those in Fig. Fig.11 into mutation rates by the method of Hall (12). The rate of lacZ GC → TA transversion was decreased by about 4.3- or 1.6-fold by MutS or MutL overexpression, respectively (Fig. (Fig.2A),2A), by a mechanism that was independent of recA function (data not shown). In contrast, the form of adaptive mutagenesis studied in most detail is strictly dependent on recA function (10, 15).

FIG. 2
MutS and to a lesser extent MutL overexpression reduced the rates of lacZ GC → TA transversion in bacterial colonies in stationary phase on plates (A) and in exponentially growing liquid cultures (B). (A) Rates of mutation were determined by the ...

The rate of lacZ GC → TA transversion was determined by the method of Lea and Coulson (17) in cells growing exponentially at 30°C in minimal A salts medium containing 0.3% glycerol (Fig. (Fig.2B).2B). MutS overexpression reduced the rate of lacZ GC → TA transversion by 2.9-fold, whereas the smaller 1.4-fold reduction by MutL overexpression was statistically marginal. Consistent with Hall's results (12), the mutation rate of the CC104(pVector) strain was about sixfold higher in stationary-phase cells in the papillation assay than in exponentially growing cells. We obtained essentially the same rate of GC → TA transversion for exponentially growing CC104 by using the Lea and Coulson method as Hall did using the Mutants C method (Fig. (Fig.2B)2B) (12).

To examine whether down regulation of the amount of MutS might contribute to the increase in GC → TA transversion in E. coli CC104, we determined the mutation rates of CC104 hfq-1 and CC104 hfq-2 mutants, which contain inactive or active Hfq protein, respectively (31, 32). We showed previously that the amount of MutS did not decrease in stationary-phase cultures of hfq-1 mutants grown overnight (31). We did not detect a significant difference in the rate of lacZ GC → TA transversion in CC104 hfq+ and either hfq mutant (data not shown). However, the hfq-1 mutants exhibit defective rpoS expression (31), and CC104 hfq-1 showed a significant lag in the formation of papillae and in growth yield in these experiments, which lasted for several days (Fig. (Fig.1).1). Therefore, while not completely conclusive, these experiments suggest that down regulation of MutS amount may not play a major role in the accumulation of GC → TA transversion mutations under these physiological conditions. Consistent with this interpretation, MutS overexpression did not influence the rate of frameshift mutation of CC107, which is very sensitive to defects in mismatch repair (8), at least in this papillation assay (see above).

One mechanism, but certainly not the only possible mechanism, for the increased GC → TA transversion in E. coli CC104 in the papillation assay is oxidative damage of guanine bases to 8-oxoguanine (8-oxoG) (20, 21, 23). 8-oxoG, which can mispair with adenine residues in DNA and seems to accumulate in nongrowing bacteria (6), is repaired in E. coli by the GO repair pathway that includes the MutY and MutM DNA glycosylases (1, 19, 20, 21, 24). We tested whether MutS or MutL overexpression could suppress lacZ GC → TA transversion in mutY or mutM mutants, which are thought to accumulate GC → TA transversions through uncorrected A:8-oxoG mismatches (1, 24). Overexpression of MutS, but not MutL, decreased the rate of lacZ GC → TA transversion in a mutY mutant by 3.4-fold to the level in the CC104 mutY+(pVector) strain (Fig. (Fig.3).3). Overexpression of MutS also significantly decreased the frequency of lacZ GC → TA transversion in a mutM mutant as judged by visual inspection of formation of papillae (data not shown). These results suggest that MutS can recognize and lead to the correction of A:8-oxoG mismatches in vivo. Consistent with this hypothesis, we found that mutL or mutS mutants have a higher rate of lacZ GC → TA transversion than their parent in this papillation assay (Fig. (Fig.4).4).

FIG. 3
MutS but not MutL overexpression decreased the rate of GC → TA transversion in an E. coli CC104 mutY mutant defective in GO repair. CC104 mutY::miniTn10(Tetr) was constructed by generalized transduction with bacteriophage P1vir. Mutation rates ...
FIG. 4
The rate of GC → TA transversion increased in mutS and mutL mutants of E. coli CC104. CC104 mutL::Ω(Kmr; BsaAI) and CC104 mutS::Ω(Kmr; EcoRV) were constructed by generalized transduction with bacteriophage P1vir (31). Mutation ...

Taken together, the results reported here suggest that MutS-dependent repair can recognize and lead to the correction of A:8-oxoG mismatches in vivo in E. coli K-12. A role for MutS-dependent mismatch repair could account for the increase in GC → TA transversion observed in mutL or mutS mutants (Fig. (Fig.4).4). Consistent with our findings, it was recently reported that the Saccharomyces cerevisiae MSH2-MSH6 heterodimer, which is a homologue of E. coli MutS, binds to A:8-oxoG mismatches in vitro and likely corrects these lesions in vivo (25). Moreover, E. coli MutS was recently reported to bind to mismatches between 5-formyluracil, which is formed by oxidative damage of thymine, and guanine residues in DNA (30). In the latter study, it was mentioned that MutS did not seem to bind appreciably to A:8-oxoG mismatches in vitro (30). Our in vivo results suggest that this biochemical issue needs to be reappraised and that MutS-dependent repair may play a general role in the correction of mismatches that arise when bases are oxidatively damaged in bacteria and yeast.

Finally, we observed by visual inspection that MutS overexpression did not reduce the frequency of AT → CG transversion in an E. coli CC101 mutT mutant (data not shown). MutT participates in GO repair by converting 8-oxoGTP to 8-oxoGMP and thereby reducing 8-oxoGTP mispairing with adenine bases in DNA during replication (20). In mutT mutants, AT → CG transversions result from MutY removing adenines of 8-oxoG:A mismatches arising from 8-oxoGTP incorporation (20, 33). The lack of effect of MutS overexpression on AT → CG transversion in a mutT mutant could simply reflect more efficient mutation fixation of 8-oxoG:A mismatches by MutY than repair by a MutS-mediated pathway, which would not be detected as a AT → CG transversion. Alternatively, MutS-mediated repair may correct A:8-oxoG mismatches that arise in mutY and mutM mutants more efficiently than it corrects 8-oxoG:A mismatches that arise in mutT mutants, where the first base in each pair is contained in a newly replicated DNA strand. As a precedent, a strand-specific mechanism mediated by MutS homologs to correct oxidatively damaged bases in human cells has been proposed (16).


We thank Patricia Foster and Tiffany Tsui for helpful information and critical discussions of this work and Jeffrey Miller for providing strains and Susan Rosenberg for providing plasmids.

This work was supported by NIH grant (RO1-CA77193) and by resources at the Lilly Research Laboratories.


1. Au K G, Cabrera M, Miller J H, Modrich P. Escherichia coli mutY gene product is required for specific A:G to C:G mismatch correction. Proc Natl Acad Sci USA. 1988;85:9163–9166. [PMC free article] [PubMed]
2. Au K G, Welsh K, Modrich P. Initiation of methyl-directed mismatch repair. J Biol Chem. 1992;267:12142–12148. [PubMed]
3. Ban C, Junop M, Yang W. Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair. Cell. 1999;97:85–97. [PubMed]
4. Biswas I, Ban C, Fleming K G, Qin J, Lary J W, Yphantis D A, Yang W, Hsieh P. Oligomerization of a MutS mismatch repair protein from Thermus aquaticus. J Biol Chem. 1999;274:23673–23678. [PubMed]
5. Bregeon D, Matic I, Radman M, Taddei F. Inefficient mismatch repair: genetic defects and down regulation. J Genet. 1999;78:21–28.
6. Bridges B A, Sekiguchi M, Tajiri T. Effect of mutY and mutM/fpg-1 mutations on starvation-associated mutation in Escherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine. Mol Gen Genet. 1996;251:352–357. [PubMed]
7. Carraway M, Marinus M G. Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli. J Bacteriol. 1993;175:3972–3980. [PMC free article] [PubMed]
8. Cupples C G, Cabrera M, Cruz C, Miller J H. A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshift mutations. Genetics. 1990;125:275–280. [PMC free article] [PubMed]
9. Feng G, Tsui H C, Winkler M E. Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells. J Bacteriol. 1996;178:2388–2396. [PMC free article] [PubMed]
10. Foster P L. Mechanisms of stationary phase mutation: a decade of adaptive mutation. Annu Rev Genet. 1999;33:57–88. [PMC free article] [PubMed]
11. Grilley M, Welsh K M, Su S S, Modrich P. Isolation and characterization of the Escherichia coli mutL gene product. J Biol Chem. 1989;264:1000–1004. [PubMed]
12. Hall B G. Spectrum of mutations that occur under selective and nonselective conditions in E. coli. Genetica. 1991;84:73–76. [PubMed]
13. Hall M C, Jordan J R, Matson S W. Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD. EMBO J. 1998;17:1535–1541. [PMC free article] [PubMed]
14. Hall M C, Matson S W. The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutH-associated endonuclease activity. J Biol Chem. 1999;274:1306–1312. [PubMed]
15. Harris R S, Feng G, Ross K J, Sidhu R, Thulin C, Longerich S, Szigety S K, Winkler M E, Rosenberg S M. Mismatch repair protein MutL becomes limiting during stationary-phase mutation. Genes Dev. 1997;11:2426–2437. [PMC free article] [PubMed]
16. Hazra T K, Izumi T, Maidt L, Floyd R A, Mitra S. The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation. Nucleic Acids Res. 1998;26:5116–5122. [PMC free article] [PubMed]
17. Lea D E, Coulson C A. The distribution of the numbers of mutants in bacterial populations. J Genet. 1949;49:264–285. [PubMed]
18. Lieb M, Bhagwat A S. Very short patch repair: reducing the cost of cytosine methylation. Mol Microbiol. 1996;20:467–473. [PubMed]
19. Michaels M L, Cruz C, Grollman A P, Miller J H. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci USA. 1992;89:7022–7025. [PMC free article] [PubMed]
20. Michaels M L, Miller J H. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) J Bacteriol. 1992;174:6321–6325. [PMC free article] [PubMed]
21. Michaels M L, Tchou J, Grollman A P, Miller J H. A repair system for 8-oxo-7,8-dihydrodeoxyguanine. Biochemistry. 1992;31:10964–10968. [PubMed]
22. Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem. 1996;65:101–133. [PubMed]
23. Moriya M, Grollman A P. Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G:C→T:A transversions induced by a single 8-oxoguanine residue in single-stranded DNA. Mol Gen Genet. 1993;239:72–76. [PubMed]
24. Nghiem Y, Cabrera M, Cupples C G, Miller J H. The mutY gene: a mutator locus in Escherichia coli that generates G:C→T:A transversions. Proc Natl Acad Sci USA. 1988;85:2709–2713. [PMC free article] [PubMed]
25. Ni T T, Marsischky G T, Kolodner R D. MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae. Mol Cell. 1999;4:439–444. [PubMed]
26. Parker B O, Marinus M G. Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proc Natl Acad Sci USA. 1992;89:1730–1734. [PMC free article] [PubMed]
27. Rasmussen L J, Samson L. The Escherichia coli MutS DNA mismatch binding protein specifically binds O(6)-methylguanine DNA lesions. Carcinogenesis. 1996;17:2085–2088. [PubMed]
28. Spampinato C, Modrich P. The MutL ATPase is required for mismatch repair. J Biol Chem. 2000;275:9863–9869. [PubMed]
29. Su S S, Lahue R S, Au K G, Modrich P. Mispair specificity of methyl-directed DNA mismatch correction in vitro. J Biol Chem. 1988;263:6829–6835. [PubMed]
30. Terato H, Masaoka A, Kobayashi M, Fukushima S, Ohyama Y, Yoshida M, Ide H. Enzymatic repair of 5-formyluracil. II. Mismatch formation between 5-formyluracil and guanine during DNA replication and its recognition by two proteins involved in base excision repair (AlkA) and mismatch repair (MutS) J Biol Chem. 1999;274:25144–25150. [PubMed]
31. Tsui H C, Feng G, Winkler M E. Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12. J Bacteriol. 1997;179:7476–7487. [PMC free article] [PubMed]
32. Tsui H C, Leung H C, Winkler M E. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol. 1994;13:35–49. [PubMed]
33. Vidmar J J, Cupples C G. MutY repair is mutagenic in mutT strains of Escherichia coli. Can J Microbiol. 1993;39:892–894. [PubMed]
34. Wu T H, Marinus M G. Deletion mutation analysis of the mutS gene in Escherichia coli. J Biol Chem. 1999;274:5948–5952. [PubMed]
35. Yamaguchi M, Dao V, Modrich P. MutS and MutL activate DNA helicase II in a mismatch-dependent manner. J Biol Chem. 1998;273:9197–9201. [PubMed]

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