Repair Of O6-methylguanine In DNA Research Articles

The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity.

The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.

Formation and repair of O6-methylguanine and methylation of the Ha-ras proto-oncogene by N-methyl-N-nitrosourea are not associated with mammary tumor resistance in the Copenhagen rat.

A high incidence of mammary adenocarcinomas is induced in sexually immature, female Buf/N rats by a single dose of N-methyl-N-nitrosourea (MNU). The Ha-ras gene is activated in a majority of these tumors. The Copenhagen (Cop) rat is completely resistant to mammary gland carcinogenesis by a number of carcinogens, including MNU. Here we show that MNU-treated Buf/N and Cop rats do not differ in the extent of formation and rate of repair of O6-methylguanine in DNA isolated from their mammary epithelial cells. Furthermore, we show that the transcriptional activities of the Ha-ras genes are similar in the mammary glands of Buf/N and Cop rats and that the extents of methylation by MNU of restriction fragments containing the Ha-ras gene from mammary gland DNA are not different for the two strains. Resistance of the Cop rat to mammary carcinogenesis, therefore, appears not to be due to a defect in the interaction of the carcinogen with the target DNA.

The Role of O6-Methylguanine in Human Cell Killing, Sister Chromatid Exchange Induction and Mutagenesis: A Review

O6-methylguanine (O6mG) produced in DNA by such SN1 methylating agents as N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been suggested by some to be the lesion that leads to certain biological endpoints in mammalian cells: cell killing, sister chromatid exchange (SCE) production, mutagenesis and cellular transformation. Other evidence is interpreted as inconsistent with this point of view. The finding of Karran & Williams (1985) that O6mG delivered to cells in culture resulted in the depletion of the activity of the protein responsible for repair of O6mG in DNA (O6mG-DNA methyltransferase, O6MT) provided a tool for the assessment of the role of O6mG in producing biological endpoints. In this paper we review much of the literature on human cells pertinent to this question. In addition we present our survival data obtained using the depletion technique of Karran & Williams as well as data supporting a model invoking a mismatch and excision response to O6mG proposed by Sklar & Strauss (1980). Although data linking O6mG to causation are inconclusive, it is premature to conclude that O6mG is not a lesion lethal to certain cultured cells.

Alcohol and cancer.

Alcohol and cancer.

Cell cycle-dependent modulation of O6-methylguanine-DNA methyltransferase in C3H/10T1/2 cells.

O6-methylguanine-DNA methyltransferase (MGMT) was measured in partially synchronized cultures of C3H/10T1/2 mouse embryo cells as a function of cell cycle. The degree of synchrony and progression of the cell cycle were monitored by flow cytometry. The MGMT level was significantly reduced prior to the onset of S-phase. This reduction was concomitant with the inhibition of in vivo repair of O6-methylguanine in DNA of S-phase cells as observed earlier. The recovery of the MGMT level paralleled the progression of synchronized cells into G2. S-phase cells purified by cell sorting contained approximately 15% of the MGMT present in G0 or early G1 cells. A comparison of the in vivo repair of O6-methylguanine and MGMT levels suggests that the lack of repair of O6-methylguanine in DNA of the mouse embryo cells is due only in part to a temporal loss of MGMT.

Methylated purines formed in DNA by dimethylnitrosamine in rats previously exposed to hepatotoxic and hepatocarcinogenic regimes: Effects on the repair of O6-methylguanine

Studies of mammalian systems for the repair of O6-methylguanine in DNA have revealed large differences in the capacities of tissues and cells to perform this function and in the case of rat liver it has been shown that the O6-methylguanine repair system can be stimulated by exposure to hepatotoxic and hepatocarcinogenic regimes. In this report an assessment is made of possible relationships between toxic liver injury, DNA synthesis, cell proliferation and DNA repair by treating Wistar rats with agents selected to provide differing degrees of liver involvement. The effects of long-term (20 week) treatments with acetylaminofluorene (15 mg/kg/day), quinoxaline 1,4-dioxide (10 mg/kg/day), 4-aminobiphenyl-HCl (15 mg/kg/day) and pronethalol (20 mg/kg/day) were assessed, using the same strain of animals in which the original toxicity and carcinogenicity data were obtained. Repair of O6-methylguanine produced in liver DNA by a low, non-toxic dose (2 mg/kg) of [14C]dimethylnitrosamine was increased 3-4-fold throughout the period of treatment with acetylaminofluorene, to a lesser extent by quinoxaline 1,4-dioxide and 4-aminophenyl-HCl and not at all in the case of pronethalol. No evidence was obtained to indicate a direct relationship between O6-methylguanine repair and either the induced hepatotoxicity or the ensuing increased rates of DNA synthesis which occur following exposure to these agents.

O6-methylguanine methyltransferase in rat liver.

The protein which catalyzes the repair of O6-methylguanine in DNA has been purified 3800-fold from rat liver. This protein acts as a methyltransferase, with the methyl group transferred to a protein-associated cysteine residue. From kinetic and physical studies, we conclude that the methyl group is transferred to the protein responsible for the activity, resulting in inactivation of the enzyme. The enzyme is asymmetric, with a molecular weight of approximately 18 500. Following methylation, there is an apparent aggregation of methylated proteins which is independent of the concentration of NaCl or nonionic detergent. Upon denaturation and analysis by gel electrophoresis, the aggregated methylated protein migrates as a single peak with a molecular weight of 18 900. The activity does not require any cofactors or divalent cations but is inhibited by NaCl. The activity also shows a preference for double-stranded DNA in terms of kinetics and efficiency of repair.

Repair of O6-methylguanine in DNA by demethylation is lacking in Mer- human tumor cell strains.

The ability of extracts of human tumor cells to demethylate O6-methylguanine (O6-MeG) in DNA was assayed using the synthetic DNA polymer poly(dC,dG,m6dG). Cell strains proficient in repair of O6-MeG in vivo (Mer+ phenotype) contained a methyltransferase activity while repair deficient cells (Mer- phenotype) had little or no activity. Mixing extracts of different Mer- strains did not result in the appearance of the activity. Extracts of Mer- cells did not inhibit the activity in extracts of Mer+ cells. Both Mer+ and Mer- strains contained methylnitrosourea-damage-specific endonuclease activity. The data suggest that the Mer- strains are deficient in methyltransferase and that this is the fundamental reason for their hypersensitivity to the cytotoxic effects of DNA alkylation. The activity was partially purified from a Mer+ colon carcinoma cell strain. Its kinetics parallel the repair of O6-MeG in DNA in vivo and suggest that the activity is inactivated during repair of DNA.

Differential repair of O(6)-methylguanine in DNA of rat hepatocytes and nonparenchymal cells.

Chronic administration of several chemical carcinogens to laboratory animals induces a variety of tumours which arise from specific cell populations within the liver. In the rat, diethylnitrosamine induces hepatocellular carcinomas, dimethylnitrosamine induces both angiosarcomas and hepatocellular carcinomas, vinyl chloride primarily induces angiosarcomas, and 1,2-dimethylhydrazine induces malignant haemangioendotheliomas. One of the principal mechanisms thought to be involved in initiating carcinogenesis is the alkylation of specific sites on DNA, such as the O(6) position of guanine. Previous investigations of alkylation and repair have, however, analysed DNA prepared from whole liver. This approach does not localize alkylation or repair capacity in the different cell types which give rise to neoplasia. Furthermore, although hepatocytes account for more than 90% of the liver's mass, they only comprise 60-70% of its cells. The nonparenchymal cell (NPC) population, which consists almost entirely of endothelial and Kupffer cells, accounts for the remaining 30-40% and contains 10-20% of the DNA. Therefore, we decided to investigate the alkylation and repair of O(6)-and 7-methylguanine in the target and non-target cells following oral administration of 1,2-dimethylhydrazine. We report here that although initial alkylation was slightly less in NPCs, removal of O(6)-methylguanine was significantly slower. This led to a preferential accumulation of O(6)-methylguanine in NPC 24 h after administering a second daily dose. In contrast, 7-methylguanine decreased at similar rates, resulting in a 28-fold greater O(6)-methylguanine/7-methylguanine ratio in the target cell population.

Pretreatment with acetylaminofluorene enhances the repair of O6-methylguanine in DNA.

Epidemiological and experimental evidence suggests that the interplay of environmental factors may be particularly relevant to the induction of cancer in man. We have investigated the possible interrelationships, at the level of DNA repair, of two chemically different hepatocarcinogens administered to rats. Animals were pretreated for several weeks by inclusion of 2-acetylaminofluorene (AAF) in the diet and a study was made of the capacity of liver to repair lesions subsequently introduced into DNA by a pulse of dimethylnitrosamine (DMN). Of particular interest was the repair of adducts at the O6 position of guanine as this product has been implicated as a critical reaction site for carcinogenesis by the monofunctional alkylating agents. We show here that the capacity of liver to repair O6-methylguanine in DNA is enhanced by prolonged exposure of rats to the chemically unrelated agent, AAF.