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A mutation in the MSH5 gene results in alkylation tolerance
Abstract:
DNA methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent carcinogens; their carcinogenic effect is mainly due to the effect of production of O6-methylguanine (O6 MeG) on DNA. O6 MeG is not only mutagenic but also toxic to the cell because Mer-/Mex- cells unable to remove O6 MeG are very sensitive to killing by MNNG. It has been proposed that repeated futile mismatch correction of O6 MeG-containing bp is responsible for the genotoxicity of the O6 MeG lesion and that loss of mismatch repair activity results in cellular tolerance to O6 MeG, but the hypothesis has not been proved. We used yeast as a model to test this hypothesis and found that chromosome deletion of any known nuclear mitotic mismatch repair genes, including MLH1, MSH2, MSH3, MSH6, and PMS1, did not rescue mgt1delta O6 MeG DNA repair methyltransferase-deficient cells from killing by MNNG. A large number of mgt1delta, MNNG-tolerant revertants were isolated, among which one cell line, XS-14, has been found to carry a mutated allele of the MSH5 gene. The mutation also affected spore survival but did not increase the spontaneous mutation rate. We further demonstrated that a mutated form of the MSH5 gene, msh5-14, not the msh5delta-null mutation, is responsible for the cellular tolerance to MNNG in XS-14 cells. This observation offers an alternative model that may reconcile seemingly contradictory observations of yeast and mammalian cells.
Insights
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) causes cancer by damaging DNA. Yeast studies show that mutations in the MSH5 gene, not other mismatch repair genes, confer tolerance to MNNG, offering a new model for DNA repair.
Area of Science:
- Molecular Biology
- Genetics
- Carcinogenesis
Background:
- DNA methylating agents like N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent carcinogens.
- The carcinogenic effects are primarily linked to O6-methylguanine (O6 MeG) DNA adducts.
- O6 MeG is mutagenic and toxic, particularly in mismatch repair-deficient (Mer-/Mex-) cells.
Purpose of the Study:
- To test the hypothesis that futile mismatch correction of O6 MeG lesions causes genotoxicity.
- To investigate the role of mismatch repair genes in cellular tolerance to MNNG.
- To explore alternative models for O6 MeG lesion processing in yeast and mammalian cells.
Main Methods:
- Utilized yeast as a model organism to study DNA repair mechanisms.
- Created chromosome deletions in known nuclear mitotic mismatch repair genes (MLH1, MSH2, MSH3, MSH6, PMS1).
- Isolated and characterized MNNG-tolerant revertants from mgt1delta cells, including sequencing for mutations in mismatch repair genes.
Main Results:
- Deletion of common mismatch repair genes did not rescue mgt1delta cells from MNNG-induced killing.
- A specific MNNG-tolerant cell line (XS-14) was found to carry a mutated allele of the MSH5 gene (msh5-14).
- The msh5-14 mutation, not a null mutation, conferred MNNG tolerance and affected spore survival without increasing spontaneous mutation rates.
Conclusions:
- Loss of mismatch repair activity does not universally confer tolerance to O6 MeG lesions.
- A specific mutation in MSH5 (msh5-14) is responsible for MNNG tolerance in yeast, challenging previous hypotheses.
- This finding provides an alternative model that may reconcile conflicting observations between yeast and mammalian cells regarding O6 MeG repair.