A system in mouse liver for the repair of O⁶-methylguanine lesions in methylated DNA

Abstract

An activity from mouse liver with catalyzes the disappearance of O6-methylguanine from DNA methylated with methylnitrosourea has been partially purified by ammonium sulfate fractionation and DNA-cellulose chromatography. The activity does not require divalent metal ions and is not affected by EDTA. It is specific for the repair of O6-methylguanine lesions and does not affect the removal of 7-methylguanine, 7-methyladenine or 3-methyladenine. The disappearance of O6-methylguanine is linear with respect to the concentration of protein and is dependent on incubation temperature. The kinetics and substrate dependence experiments suggest that the protein factor is product-inactivated. Amino acid analysis of hydrolysates of protein obtained after incubation of methylated DNA with the protein factor indicates the presence of radiolabeled S-methyl-L-cysteine, suggesting that during the repair of O6-methylguanine from methylated DNA, the methyl group is transferred to a sulfhydryl of a cysteine residue of a protein. This represents the first such demonstration in a mammalian system.

Tamoxifen accelerates proteasomal degradation of O6‐methylguanine DNA methyltransferase in human cancer cells

“…7,8 This action inactivates one MGMT protein molecule for each lesion repaired and makes MGMT a suicide protein, because alkylated MGMT is then degraded through the ubiquitin-dependent proteasomal pathway. 9 The alkylated base adduct is generated in DNA either endogenously or after exposure to alkylating carcinogens and antitumor drugs with methylating and chloroethylating properties, such as chemotherapeutic 2-chloroethyl-N-nitrosourea (CNU) derivatives [e.g., 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] 8,10 and monofunctional triazenes [e.g., dacarbazine (DTIC)].…”

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

“…In human cells or cell extracts at 37 ° the half-life was about 10 min or less Yarosh et al, 1983;Foote et al, 1983;Harris et al, 1983). The reaction was equally as rapid with the rodent O6-MT (Bogden et al, 1981;Scicchitano and Pegg, 1982). Once the O6-MT in a cell has reacted, only RNA and protein synthesis restored the original levels of 06-MT (Robins and Cairns, 1979;Warren and Lawley, 1980;Waldstein et al, 1982b;Yarosh et al, 1984a), demonstrating that the O6-MT is not rejuvenated after its reaction with a methyl group, and remains as a dead end complex.…”

“…This difference may be resolved when the human O6-MT is available in pure form. The bacterial O6-MT was less active but still functional at 5 ° or less (Foote et al, 1980;Lindahl et al, 1982) while the mammalian O6-MT was virtually inactive at these temperatures (Bogden et al, 1981;.…”

“…DNA-repair activities analogous to E. coli 0 6-MT were soon identified in mouse liver (Bogden et al, 1981), rat liver (Renard and Verly, 1980;Mehta et al, 1981), human liver , and these activities were present in human cells in culture (Waldstein et al, 1982a;Myrnes et al, 1982;Foote et al, 1983;Yarosh et al, 1983a, b). Evidence for an adaptive response in mammalian cells which paralleled the E. eoli response was more ambiguous.…”

“…In human fibroblasts ethylphosphotriesters were not removed, O4-ethylthymine (O4-EtThy) had a half-life of 4-5 days, but 0 6-EtGua was removed with a half-life of less than 20 h (Bodell et al, 1979). C57/BL mice treated with MNU or ENU removed very little alkylphosphotriesters from the DNA of many organs including liver although O6-MT was present in C57/BL primary tissue and cells in culture (Yagi et al, 1984) and has been partially purified from the liver of this strain of mouse (Bogden et al, 1981). In rat liver O4-alkylThy was removed very slowly, with a half-life of 4 days or longer, and alkylphosphotriesters were essentially not repaired, while O6-alkylGua had a half-life of 13 h or less (O'Connor et al, 1973;Scherer et al, 1980;Singer et al, 1981;Swenberg et al, 1984).…”