Removal of O6-methylguanine from DNA by human liver fractions.

Abstract

In in vitro assays using methylated DNAs as substrates, human liver fractions were shown to be able to catalyze the removal of 06-methylguanine. The amount of removal was proportional to the amount of protein added, and the loss of O6-methylguanine occurred with stoichiometric formation of guanine in the DNA and S-methylcysteine in protein. This indicates that human liver contains a protein similar to that previously found in bacteria exposed to ailcylating agents. This protein acts as a transmethylase, transferring the intact methyl group from 06-methylguanine in DNA to a cysteine residue on that protein. A similar activity is present in rodent liver, butit was found that human liver was about 10 times more active in carrying out this reaction. In contrast, there was no difference between the human and rat liver extracts in catalyzing the loss of another methylation product, 7-methylguanine, from alkylated DNA. The liver is the organ most likely to be alkylated after exposure to exogenous potential alkylating agents such as dimethylnitrosamine. The present results show that human liver has a significant capacity to repair 06-methylguanine in DNA, which has been implicated as a critical product in carcinogenesis and mutagenesis. Dimethylnitrosamine (Me2NNO) is known to be carcinogenic in many animal species (1). This effect has been associated with the capacity of the target tissues to metabolize Me2NNO into a mutagenic intermediate that reacts with DNA at various sites (2-4). Of the various DNA adducts formed, 06-methylguanine (MeGua) has been implicated in both mutagenesis and carcinogenesis (4-10). The persistence of06-alkylguanine in the DNA of a tissue correlates with the probability that that tissue will develop tumors after administration of alkylating agents, indicating that this adduct is a critical determinant in the initiation of carcinogenesis by nitrosamines such as Me2NNO (4, 9, 10). MeGua can be removed from DNA in Escherichia coli by a DNA repair system which transfers the methyl groups to a cysteine residue in protein (11,12). There is evidence that this system also occurs in rodent tissues, but it has not been fully characterized (4,(13)(14)(15)(16)(17). Humans are known to be exposed to nitrosamines, including Me2NNO (18,19), and Me2NNO is metabolized by human liver to generate the reactive intermediate that results in DNA alkylation (20,21).In the work presented here, we used an in vitro assay to demonstrate that human liver fractions can catalyze the removal of MeGua from DNA; we found that this capacity is about 10 times greater than that with comparable rat liver fractions. We have also shown that this human liver system transfers the methyl group from the 06 position of guanine to eysteine residue, regenerating guanine directly in the DNA. MATERIALS AND METHODS Chemicals. N-[3H]Methyl-N-nitrosourea (1.6 Ci/mmol; I Ci = 3.7 X 1010 becquerels) was obtained from New England Nuclear. All other biochemical reagents were obtained from Sigma.Tissues. The 10 samples of human liver (six m...

The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis

“…Transfer of the methyl group produced an SMeCys residue in both bacterial and mammalian O6-MT (Olsson and Lindahl, 1980;Bogden et al, 1981;Mehta et al, 1981;Pegg et al, 1982). Loss of O6-MeGua from DNA was coupled with a stoichiometric formation of S-MeCys Bogden et al, 1981;Pegg et al, 1982;Craddock et al, 1982;Waldstein et al, 1983), indicating that this is the exclusive mechanism for demethylation of O6-MeGua.…”

“…Loss of O6-MeGua from DNA was coupled with a stoichiometric formation of S-MeCys Bogden et al, 1981;Pegg et al, 1982;Craddock et al, 1982;Waldstein et al, 1983), indicating that this is the exclusive mechanism for demethylation of O6-MeGua. Purification of the E. coli O6-MT has demonstrated that the transferring and accepting functions reside on the same protein and that a single O6-MT molecule can act only once Lindahl et al, 1982).…”

“…Unadapted E. coli contained very low levels of O6-MT, approximately 13-60 molecules per cell (Mitra et al, 1982), and unadapted B. subtilis had about 240 molecules per cell (Hadden et al, 1983;Morohoshi and Munkata, 1983). Normal human fibroblasts contained 40000-80000 molecules per cell (Metcalf and Lawley, 1981), human lymphoid cells had 10000-25000 per cell and human tumor cells had between 30000 and 100000 per cell (Yarosh et al, 1983a;Foote et ai., 1983;Foote and Mitra, 1984;Myrnes et al, 1982;Waldstein et al, 1982a).…”

“…Many of the Ada-mutants isolated so far appear to lack both the repair and regulatory functions of the induced O6-MT protein. They contained a normal basal level of O6-MT activity (Mitra et al, 1982). Upon adaption they produced a 37-kd protein which cross-reacted with the anti-O6-MT antibody (Teo et al, 1984) but no O6-MT activity was detected (Jeggo, 1979;Karran et al, 1979;Mitra et al, 1982) and the alk gene product was not induced (Evensen and Seeberg, 1982).…”

“…Upon adaption they produced a 37-kd protein which cross-reacted with the anti-O6-MT antibody (Teo et al, 1984) but no O6-MT activity was detected (Jeggo, 1979;Karran et al, 1979;Mitra et al, 1982) and the alk gene product was not induced (Evensen and Seeberg, 1982). Another class of Ada mutants (Adc) overproduced, without adaption, O6-MT indistinguishable from wild-type but did not overproduce the glycosylase, and 'reverted' to the Ada-phenotype (Sedgwick and Robins, 1980;Mitra et al, 1982;Demple et al, 1982;Karran et al, 1982a).…”