A single amino acid substitution in MSH5 results in DNA alkylation tolerance
Introduction
O6-Methylguanine DNA methyltransferase (O6-MeG MTase) activity has been found in all organisms examined to date and is considered primarily responsible for repairing DNA lesions induced by SN1-type alkylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (Pegg, 1990). Mammalian cells lacking O6-MeG MTase also display enhanced sensitivity to killing by chemotherapeutic bifunctional nitrosoureas such as carmustine, lomustine and streptosotocin Pegg, 1990, Karran and Hampson, 1996. There have been several independent reports that mutations in mammalian mismatch repair genes such as hMSH2 (Aquilina et al., 1998), hMSH6 Kat et al., 1993, Papadopoulos et al., 1995, Hampson et al., 1997, hMLH1 Koi et al., 1994, Hampson et al., 1997 and hPMS2 Risinger et al., 1995, Ciotta et al., 1998 may be responsible for rescuing MTase-deficient cells from killing by DNA alkylating agents. Furthermore, at least two mouse mismatch repair-deficient models have been examined with respect to alkylation tolerance de Wind et al., 1995, Kawate et al., 1998, and a link was established between a mismatch repair defect and alkylation tolerance. An abortive mismatch repair hypothesis was initially proposed by Goldmacher et al. (1986) and then by Branch et al. (1993) to explain the observed phenomena. However, an alternative hypothesis of mismatch repair-induced signaling for apoptosis also gained recent experimental support D'Atri et al., 1998, Hickman and Samson, 1999.
The DNA mismatch repair system is conserved throughout living organisms. In particular, eukaryotic cells from yeast to human appear to contain a similar set of mismatch repair genes to the extent that each mismatch repair gene in human has a structural and functional homolog in a yeast cell. Our laboratory used Saccharomyces cerevisiae as a model lower eukaryote and found that deletion of any major mismatch repair genes, including MSH2, MSH3, MSH6, MLH1 and PMS1, in the mgt1Δ mutant deficient in MTase activity (Xiao et al., 1991) did not rescue it from killing by MNNG (Xiao et al., 1995). Subsequently, we mutagenized mgt1Δ cells, isolated MNNG-tolerant mutants and characterized one of these recessive mutants, XS-14 (Bawa and Xiao, 1997). Transformation of XS-14 cells with each wild-type mismatch repair gene did not alter its alkylation tolerance. Surprisingly, transformation of XS-14 cells with a single-copy MSH2 homolog MSH5 can strongly suppress the MNNG-tolerant phenotype, although deletion of the MSH5 gene did not result in alkylation tolerance. Through genetic analysis, the mutation in XS-14 was mapped within the MSH5 locus (Bawa and Xiao, 1997). Here we describe our investigation of various msh5 alleles and demonstrate that a single mutation within the MSH5 gene that results in an amino acid substitution is responsible for the observed alkylation tolerance.
Section snippets
Yeast strains and transformation
Yeast strains used in this study are listed in Table 1. XS-803-2C was obtained from Dr. D. Gietz (University of Manitoba, Canada). The creation of its mgt1Δ::LEU2 and msh5Δ::hisG derivatives and the isolation of XS-14 have been described previously (Bawa and Xiao, 1997). Yeast cells were grown in either complete YPD (1% yeast extract, 2% bacto-peptone, 2% glucose) or synthetic SD (0.67% yeast extract without nitrogen bases, 2% glucose) supplemented with amino acids and bases as recommended
msh5-14 contains a single point mutation
In order to understand the mechanism by which a point mutation in MSH5 results in alkylation tolerance, we isolated the mutated MSH5 allele, msh5-14, from XS-14 and determined the entire nucleotide sequence of the msh5-14 open reading frame plus a 266-bp promoter region and compared it with the published MSH5 sequence (Hollingsworth et al., 1995). To our surprise, as many as six nucleotide sequence variations (T-28C, C1299T, A1361G, G1494T, C1570T and T2467C, relative to the MSH5 translation
Discussion
We report here the detailed characterization of the msh5-14 mutation that results in an increased alkylation tolerance. We found a single de novo mutation in the msh5-14 allele along with several preexisting sequence variations. We demonstrated that the resulting Y823H single amino acid substitution is sufficient to cause alkylation tolerant phenotypes. It is interesting to note that the Y823H substitution falls in a well-conserved region known as a helix-u-turn-helix (H-U-H) motif (Hsieh, 2001)
Acknowledgements
We thank Drs. D. Gietz for the yeast strain, N. Hollingsworth for the MSH5 gene and L. Worth for helpful discussion. We also thank M. Hanna for proofreading the manuscript. This research was supported by the Canadian Institutes of Health Research operating grant MOP-38104 (to W.X.).
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