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Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency.

Taira K, Nakamura S, Nakano K, Maehara D, Okamoto K, Arimoto S, Loakes D, Worth L, Schaaper RM, Seio K, Sekine M, Negishi K, Negishi T - Mutat. Res. (2007)

Bottom Line: Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis.Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C.These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.

ABSTRACT
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O(6)-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O(6)-MeG:T, O(6)-MeG:C, O(6)-EtG:T, O(6)-EtG:C, G:T and G:C. Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C. These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.

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The mutational frequencies of MNU (A), MNNG (B), MMS (C), ENU (D), ENNG (E) and EMS (F) in a MutS-deficient strain (filled circle) or wild-type (open circle). *P < 0.05, **P < 0.01 compared to the mutant frequency for the wild-type. Three to six independent experiments were performed for each. Mutational frequencies were obtained after correcting for the frequency of spontaneous mutations. The frequencies of spontaneous mutations are (34.3 ± 3.3) × 10−7 for the MutS (−) strain (NR12986) and (0.3 ± 0.1) × 10−7 for the wild-type strain (NR10832).
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fig2: The mutational frequencies of MNU (A), MNNG (B), MMS (C), ENU (D), ENNG (E) and EMS (F) in a MutS-deficient strain (filled circle) or wild-type (open circle). *P < 0.05, **P < 0.01 compared to the mutant frequency for the wild-type. Three to six independent experiments were performed for each. Mutational frequencies were obtained after correcting for the frequency of spontaneous mutations. The frequencies of spontaneous mutations are (34.3 ± 3.3) × 10−7 for the MutS (−) strain (NR12986) and (0.3 ± 0.1) × 10−7 for the wild-type strain (NR10832).

Mentions: To evaluate the involvement of MMR in protection against mutagenesis induced by DNA methylation and ethylation, the mutagenicity of MNU, MNNG, MMS, ENU, ENNG or EMS was measured in repair proficient- and MutS-deficient E. coli CC102 cells, in which GC to AT reversions can be detected. For both strains, the mutational frequency increased with an increase in the concentration of each alkylating agent applied to the cells. The mutational frequencies for MNU, MNNG and MMS were elevated in the MutS-deficient strain more sharply than in wild-type (Fig. 2A, B and C). In contrast, the mutational frequencies induced by ENU and ENNG were almost the same between the strains (Fig. 2D and E). The frequencies of mutation by EMS were significantly higher in MutS (−) than that in wild-type, but the differences were very small. Treatment of cells with mutagens at these concentrations did not result in significant levels of cell death.


Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency.

Taira K, Nakamura S, Nakano K, Maehara D, Okamoto K, Arimoto S, Loakes D, Worth L, Schaaper RM, Seio K, Sekine M, Negishi K, Negishi T - Mutat. Res. (2007)

The mutational frequencies of MNU (A), MNNG (B), MMS (C), ENU (D), ENNG (E) and EMS (F) in a MutS-deficient strain (filled circle) or wild-type (open circle). *P < 0.05, **P < 0.01 compared to the mutant frequency for the wild-type. Three to six independent experiments were performed for each. Mutational frequencies were obtained after correcting for the frequency of spontaneous mutations. The frequencies of spontaneous mutations are (34.3 ± 3.3) × 10−7 for the MutS (−) strain (NR12986) and (0.3 ± 0.1) × 10−7 for the wild-type strain (NR10832).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2365708&req=5

fig2: The mutational frequencies of MNU (A), MNNG (B), MMS (C), ENU (D), ENNG (E) and EMS (F) in a MutS-deficient strain (filled circle) or wild-type (open circle). *P < 0.05, **P < 0.01 compared to the mutant frequency for the wild-type. Three to six independent experiments were performed for each. Mutational frequencies were obtained after correcting for the frequency of spontaneous mutations. The frequencies of spontaneous mutations are (34.3 ± 3.3) × 10−7 for the MutS (−) strain (NR12986) and (0.3 ± 0.1) × 10−7 for the wild-type strain (NR10832).
Mentions: To evaluate the involvement of MMR in protection against mutagenesis induced by DNA methylation and ethylation, the mutagenicity of MNU, MNNG, MMS, ENU, ENNG or EMS was measured in repair proficient- and MutS-deficient E. coli CC102 cells, in which GC to AT reversions can be detected. For both strains, the mutational frequency increased with an increase in the concentration of each alkylating agent applied to the cells. The mutational frequencies for MNU, MNNG and MMS were elevated in the MutS-deficient strain more sharply than in wild-type (Fig. 2A, B and C). In contrast, the mutational frequencies induced by ENU and ENNG were almost the same between the strains (Fig. 2D and E). The frequencies of mutation by EMS were significantly higher in MutS (−) than that in wild-type, but the differences were very small. Treatment of cells with mutagens at these concentrations did not result in significant levels of cell death.

Bottom Line: Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis.Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C.These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.

ABSTRACT
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O(6)-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O(6)-MeG:T, O(6)-MeG:C, O(6)-EtG:T, O(6)-EtG:C, G:T and G:C. Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C. These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.

Show MeSH
Related in: MedlinePlus