The repair of specific types of DNA alkylation damage by O6-alkylguanine-DNA alkyltransferase (MGMT) is a major mechanism of resistance to the carcinogenic and chemotherapeutic effects of certain alkylating agents. MGMT expression levels vary widely between individuals but the underlying causes of this variability are not known. To address this, we used an expressed single nucleotide polymorphism (SNP) and demonstrated that the MGMT alleles are frequently expressed at different levels in peripheral blood mononuclear cells (PBMC). This suggests that there is a genetic component of inter-allelic variation of MGMT levels that maps close to or within the MGMT locus. We then used quantitative trait locus (QTL) analysis using intragenic SNPs and found that there are at least two sites influencing inter-individual variation in PBMC MGMT activity. One is characterized by an SNP at the 3' end of the first intron and the second by two SNPs in the last exon. The latter are in perfect disequilibrium and both result in amino acid substitutions-one of them, Ile143Val, affecting an amino acid close to the Cys145 residue at the active site of MGMT. Using in vitro assays, we further showed that while the Val143 variant did not affect the activity of the protein on methylated DNA substrate, it was more resistant to inactivation by the MGMT pseudosubstrate, O6-(4-bromothenyl)guanine. These findings suggest that further investigations of the potential epidemiological and clinical significance of inherited differences in MGMT expression and activity are warranted.
The alkyltransferase-like (ATL) proteins contain primary sequence motifs resembling those found in DNA repair O6-alkylguanine-DNA alkyltransferase proteins. However, in the putative active site of ATL proteins, a tryptophan (W83) residue replaces the cysteine at the known active site of alkyltransferases. The Escherichia coli atl gene was expressed as a fusion protein and purified. Neither ATL nor C83 or A83 mutants transferred [3H] from [3H]-methylated DNA to themselves, and the levels of O6-methyl guanine (O6-meG) in substrate DNA were not affected by ATL. However, ATL inhibited the transfer of methyl groups to human alkyltransferase (MGMT). Inhibition was reduced by prolonged incubation in the presence of MGMT, again suggesting that O6-meG in the substrate is not changed by ATL. Gel-shift assays show that ATL binds to short single- or double-stranded oligonucleotides containing O6-meG, but not to oligonucleotides containing 8-oxoguanine, ethenoadenine, 5-hydroxycytosine or O4-methylthymine. There was no evidence of demethylation of O6-meG or of glycosylase or endonuclease activity. Overexpression of ATL in E.coli increased, or did not affect, the toxicity of N-methyl-N′-nitro-N-nitrosoguanidine in an alklyltransferase-proficient and -deficient strain, respectively. These results suggest that ATL may act as a damage sensor that flags O6-meG and possibly other O6-alkylation lesions for processing by other repair pathways.
Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.
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