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Biomedical subjects

M E Dolan

Publications and source records attributed to M E Dolan.

At least 91 records · Page 5Linked to original sources
PubMed1991

Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts.

Antisera were raised in rabbits to three peptides which correspond to sequences of amino acids present in human O6-alkylguanine-DNA alkyltransferase (residues 1-11, 8-20 and 197-207). These antisera recognized the alkyltransferase protein on Western blots of proteins from a number of Mer+ human tumor cell lines but this protein was found to be absent from Mer- tumor cell lines. The alkyltransferase protein was also detected by these antisera in some extracts from human liver samples but other human liver extracts having a high alkyltransferase activity failed to react with antibodies directed towards the carboxyl terminus of the protein, suggesting that part of this region can be removed by protease action without loss of activity or that there is genetic variability in this region. This result indicates that studies with a number of antisera or with an antibody known to be directed towards an essential, invariant region of the alkyltransferase will be needed for unequivocal detection of the alkyltransferase by antibody screening. The immunoreactive human alkyltransferase protein was absent from CHO cell lines that had previously been selected for alkyltransferase expression after transfection with human DNA. It is therefore probable that the hamster gene has been reactivated in these cells.

Animals↗
PubMed1991

Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine.

Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in human O6-alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure to N-methyl-N'-nitro-N-nitrosoguanidine or to O6-benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine respectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any significant extent. The short half-life of the inactivated alkyltransferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repair O6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.

Amino Acid Sequence↗
PubMed1990

Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents.

O6-Alkylguanine-DNA alkyltransferase was rapidly and irreversibly inactivated by exposure to O6-benzylguanine or the p-chlorobenzyl and p-methylbenzyl analogues. This inactivation was much more rapid than with O6-methylguanine: incubation with 2.5 microM O6-benzylguanine led to more than a 90% loss of activity within 10 min, whereas 0.2 mM O6-methylguanine for 60 min was required for the same reduction. O6-Benzylguanine was highly effective in depleting the alkyltransferase activity of cultured human colon tumor (HT29) cells. Complete loss of activity was produced within 15 min after addition of O6-benzylguanine to the culture medium and a maximal effect was obtained with 5 microM. In contrast, at least 100 microM O6-methylguanine for 4 hr was needed to get a maximal effect, and this reduced the alkyltransferase by only 80%. Pretreatment of HT29 cells with 10 microM O6-benzylguanine for 2 hr led to a dramatic increase in the cytotoxicity produced by the chemotherapeutic agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-chloroethyl(methysulfonyl)methanesulfonate (Clomesone). Administration of O6-benzylguanine to mice at a dose of 10 mg/kg reduced alkyltransferase levels by more than 95% in both liver and kidney. These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents.

Antineoplastic Agents↗
PubMed1990

Modulation of mammalian O6-alkylguanine-DNA alkyltransferase in vivo by O6-benzylguanine and its effect on the sensitivity of a human glioma tumor to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea.

Experiments were carried out in mice and hamsters to determine whether the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, in tissues and tumors was reduced by treatment with O6-benzylguanine in vivo. Following intraperitoneal injection of O6-benzylguanine, there was a rapid and complete loss of alkyltransferase activity in both livers and kidneys of mice and hamsters. The activity in mouse tissues was slowly restored, reaching pretreatment activities at 16 hr and 72 hr after injection of O6-benzylguanine at 10 mg/kg or 126 mg/kg, respectively. The activity in hamster liver was restored at a significantly lower rate, reaching less than 20% pretreatment activity 72 hr after treatment with 100 mg/kg of O6-benzylguanine. The efficient reduction of alkyltransferase activity by O6-benzylguanine was in sharp contrast to the inability of O6-methylguanine to bring about similar reductions. Activities dropped to about 55% of pretreatment activities in several mouse organs 4 hr after treatment with 126 mg/kg of O6-methylguanine compared to a more than 90% reduction in activity in animals after treatment with O6-benzylguanine. The sensitivity of SF767 cells to meCCNU after treatment with O6-benzylguanine was increased substantially. Furthermore, treatment of nude mice carrying SF767 tumor with 60 mg/kg of O6-benzylguanine prior to either 7.5 or 15 mg/kg of meCCNU led to significant inhibition of tumor growth. These studies indicate that O6-benzylguanine is a suitable compound for use in experiments to examine the role of the alkyltransferase protein in vivo in counteracting the effects of alkylating agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Depletion of O6-alkylguanine-DNA alkyltransferase activity in mammalian tissues and human tumor xenografts in nude mice by treatment with O6-methylguanine.

We have previously shown that exposure of cells in culture to O6-methylguanine significantly reduces their level of the repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), thus rendering cells more sensitive to the cytotoxic effects of chemotherapeutic chloroethylating agents. Experiments were carried out in mice to determine whether the AGT content of tissues and tumors could be reduced by in vivo treatment with O6-methylguanine. There was a dose-dependent decrease in AGT activity in liver tissues of CD-1 mice to 24% of basal levels after four hourly intraperitoneal injections of O6-methylguanine (110 mg/kg). Although the decline in AGT activity in the liver was reversible, the activity remained at 75% of basal levels for up to 25 h after the final injection. The effect of O6-methylguanine treatment on AGT activity was measured in mouse tissues as well as human colonic carcinoma tumors (HT29 and BE) grown in Swiss athymic nude mice. The activity in the liver, kidney, and spleen of these mice decreased to 33%-35% of control levels, whereas the activity in HT29 tumors was likewise diminished to 25% of control levels after four hourly injections of O6-methylguanine (100 mg/kg). There was no enhancement of the tumoricidal effectiveness of chloroethylating agents on the HT29 tumor after O6-methylguanine treatment, probably due to a disproportionately higher level of AGT in human tissue than in murine tissue. However, these studies suggest that O6-methylguanine can be given in vivo to examine the role of the AGT protein in protecting against the toxic and carcinogenic effects of alkylating agents.

Animals↗
PubMed1989

Expression of mammalian O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents.

Chinese hamster ovary cells (CHO) were co-transfected with pSV2neo and sheared DNA from either a human cell line (HT29) expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) or from a cell line (BE) deficient in this activity. Cells expressing the selectable marker were obtained by exposure to G418 and colonies resistant to alkylation damage isolated by growth in the presence of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The number of colonies of cells expressing AGT activity arising after transfection with DNA from BE cells was similar to the number arising from cells exposed to HT29 DNA. Although the amount of AGT repair protein expressed in the transfectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-deficient cells by transfection of human DNA from both repair-deficient and proficient cells. A separate transfection of CHOMG cells [a mutant of CHO cells resistant to the drug, methylglyoxal bis(guanylhydrazone) (MGBG)] with HT29 DNA and pSV2neo followed by selection of G418 and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in three colonies with high AGT levels. These transfectants had different growth rates and expressed levels of the AGT protein between 230 and 300 fmol/mg protein. The transfectants were as resistant to the cytotoxic effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as HT29 cells which were much more resistant than the parental CHOMG cells. Pretreatment of transfectant cells with 0.4 mM O6-methylguanine for 24 h reduced AGT activity to 14% basal levels, which upon removal of the base increased to approximately 74% basal level within 8 h. The sensitivity to the cytotoxic effects of both the chloroethylating and methylating agents was enhanced by treatment with O6-methylguanine. In the same manner, the number of BCNU-induced DNA interstrand cross-links increased in transfectant cells pretreated with O6-methylguanine. These results provide further evidence that the formation of methyl or chloroethyl adducts at the O6-position contribute significantly to cell lethality.

Alkylating Agents↗
PubMed1988

Effect of O6-methylguanine on DNA interstrand cross-link formation by chloroethylnitrosoureas and 2-chloroethyl(methylsulfonyl)methanesulfonate.

Exposure of HT29 cells in culture to O6-methylguanine is known to result in a reduction in O6-alkylguanine-DNA alkyltransferase (AGT) activity and an enhancement of sensitivity to the cytotoxic effects of chloroethylating agents. Since cytotoxicity of these agents may be mediated by the formation of interstrand cross-links, alkaline elution analysis was performed on HT29 cells treated with 1-(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and Clomesone [2-chloroethyl(methylsulfonyl)methanesulfonate] in the presence or absence of O6-methylguanine pretreatment to determine if the enhanced toxicity was due to an increase in the number of cross-links formed. Interstrand cross-linking by 1-(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was increased by pretreatment with 0.4 mM O6-methylguanine for 24 h. Cross-linking by Clomesone was observed only in cells exposed to 0.4 mM O6-methylguanine for 24 h prior to administration of the drug and for 12 h after administration, suggesting that the resynthesis of the AGT may prevent the cross-linking by Clomesone. Complete recovery of AGT activity after reduction to 20 to 30% of the basal level upon treatment with 0.4 mM O6-methylguanine required between 8 h and 15 h in both HT29 cells and in Raji cells which were also sensitized to 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea by exposure to O6-methylguanine. These data suggest that the enhancement of chloroethylnitrosourea toxicity after treatment with O6-methylguanine may be related to an increase in the number of DNA cross-links and that the relatively rapid rate of AGT recovery plays a role in prevention of cross-links resulting from Clomesone.

Cell Division↗
PubMed1988

Use of oligodeoxynucleotides containing O6-alkylguanine for the assay of O6-alkylguanine-DNA-alkyltransferase activity.

A sensitive assay procedure was developed for the measurement of the activity of mammalian O6-alkylguanine-DNA-alkyltransferase. The procedure utilized oligodeoxynucleotides containing O6-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with 32P by the reaction with polynucleotide kinase and [gamma-32P]ATP and allowed to react with organ or cell extracts containing the alkyltransferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyltransferase is bimolecular, the second order rate constants for the reaction between the labeled oligomer and repair protein from several different sources were determined. The amount of alkyltransferase present was then calculated from the amount of product formed and the appropriate second order rate constant for the reaction. Excellent agreement was obtained between the alkyltransferase levels determined in this procedure and those measured by conventional assay procedures in a variety of cell lines having both high and low activity. The method also gave results in good agreement with other assay procedures for a number of rat tissues, although a few tissues gave anomalous results owing to a high level of nuclease activity which degraded the substrate. This method should prove useful for the measurement of alkyltransferase activity in samples in which the activity is very low or the amount of material available is limited.

DNA Repair↗
PubMed1988

Investigation of the specificity of O6-alkylguanine-DNA-alkyltransferase.

Comparison of the abilities of alkylated RNA and DNA to serve as substrates for the O6-alkylguanine-DNA-alkyltransferase have been carried out. It was found that the O6-methylguanine in tRNA was much less active as a substrate for the protein than O6-methylguanine in double stranded DNA. The difference in rates of repair was such that it is unlikely that the alkyltransferase would act on RNA in vivo and, therefore, the reaction with RNA should not contribute towards the exhaustion of its repair capacity.

Chromatography, High Pressure Liquid↗
PubMed1988

Use of a dodecadeoxynucleotide to study repair of the O4-methylthymine lesion.

A dodecadeoxynucleotide of defined sequence containing O4-methylthymine was labeled at the 5' end with [32P] by the reaction with (gamma-32P]ATP and polynucleotide kinase. Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion. The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient. There was complete repair of O4-methylthymine by the E. coli alkyltransferase upon incubation for 4 h at 37 degrees C. There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period. There was also no repair of the O4-methylthymine lesion in the presence of crude rat-liver extract. However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this. The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O6-alkylguanine repair.

Animals↗
PubMed1988

Repair of O-alkylpyrimidines in mammalian cells: a present consensus.

Enzymatic repair of the O-alkylpyrimidines (O2- and O4-alkylthymine, O2-alkylcytosine) and alkyl phosphotriesters has been studied in Escherichia coli, and the two proteins involved, a glycosylase (DNA-3-methyladenine glycosylase) and a methyltransferase (DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), have been well characterized. In mammals or mammalian cells treated with carcinogenic alkylating agents, loss of these derivatives has been demonstrated repeatedly. Nevertheless, mammalian repair proteins that are analogous to those from E. coli do not detectably act on these alkyl derivatives. A variety of techniques has been used by many investigators in the United States and Europe, who conclude here that the mode of O-alkylpyrimidine and alkyl phosphotriester repair in mammalian cells differs from that in E. coli. New approaches and methods are needed to characterize these processes at the biochemical and molecular level.

Alkylating Agents↗
PubMed1988

Cell specific differences in O6-methylguanine-DNA methyltransferase activity and removal of O6-methylguanine in rat pulmonary cells.

Previous studies have demonstrated that cell specificity exists for the alkylation of DNA from lung cells following treatment of rats with the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The concentration of the promutagenic adduct O6-methylguanine (O6MG) was found to be greatest in Clara cells followed by macrophages, type II cells and alveolar small cells. The purpose of this study was to measure the activity of the repair protein O6-methylguanine-DNA methyltransferase (O6MGMT) and to determine whether differences exist for the removal of O6MG among pulmonary cell types. Constitutive activity of O6MGMT was 2-fold greater in macrophages and type II cells than alveolar small cells and Clara cells. Treatment for 4 days with NNK (10 mg/kg/day) had no effect on O6MGMT activity in macrophages, but decreased activity in alveolar small cells and type II cells by 57 and 84%, respectively. O6MGMT activity was reduced to below limits of detection in Clara cells following treatment with NNK. The effect of NNK on O6MGMT activity was consistent with rates of removal of O6MG in macrophages and Clara cells. The loss of O6MG from DNA of macrophages followed first order kinetics (t1/2 = 48 h) while very little loss of this adduct was observed in Clara cells over an 8 day period following cessation of carcinogen treatment. Even though O6MGMT activity was reduced in alveolar small cells and type II cells, approximately 90% of the O6MG bound to DNA in these cell types was removed within 8 days after treatment was discontinued. The loss of O6MG from pulmonary cells appears to result largely from the removal of this adduct by O6MGMT since rates of cell turnover were very low (0.5-1.5%/day) in the lung and were not affected by treatment with NNK. This study indicates that the activity of O6MGMT and the rate of resynthesis of this repair enzyme differ considerably among pulmonary cells following the methylation of DNA. The high concentration of O6MG in Clara cells and the low rate of repair of this promutagenic adduct may be critical factors in the potent pulmonary carcinogenicity induced by the tobacco specific carcinogen NNK.

Animals↗
PubMed1988

Sequence specificity of guanine alkylation and repair.

The sequence selectivity of methylation at the O6 and N7 position of guanine by N-methyl-N'-nitrosourea (MNU) and the rate of removal of O6-methylguanine by O6alkylguanine-DNA alkyltransferase (AGT) was determined using dodecadeoxynucleotides of defined structure. The extent of guanine adduct formed in self-complementary dodecamers, 5'-TATACGCGTATA-3', 5'-TATACCGGTATA-3' and 5'-TATAGGCCTATA-3', after methylation with [3H]MNU in a representative experiment were, respectively, 10, 19 and 30 pmol O6-methylguanine/mumol guanine and 97, 189 and 217 pmol N7-methylguanine/mumol guanine. The O6-methylguanine/N7-methylguanine ratio remained relatively constant for each dodecamer. A direct comparison between the methylation at guanine with adenine or thymine as the 5'-flanking base was made with two dodecamers, 5'-TATACATGTATA-3' and 5'-TATACTAGTATA-3'. When the guanine residue was preceded 5' by an adenine, the level of O6 and N7-alkylation was, respectively, 2.1-fold and 1.5-fold greater than when guanine was preceded 5' by a thymine. These date are consistent with a regioselective mechanism for alkylnitrosourea alkylation of guanine. The methylated dodecamer, 5'-TATACGCGTATA-3' was repaired faster than 5'-TATACCGGTATA-3' by HT29 extract containing AGT with a loss in 10 min of 0.052 pmol and 0.025 pmol O6-methylguanine, respectively. Dodecamers of the structure 5'-dCGCGAATTCm6GCG-3' and 5'-dCGCCAATTGm6GCG-3' were labeled at the 5' end with 32P by the reaction with polynucleotide kinase and after incubation with AGT, the methylated and demethylated dodecamers were separated by reversed-phase HPLC. The amount of demethylated product formed was greater for the dodecamer containing cytosine as the 5'-flanking base to O6-methylguanine compared to guanine in that same position. A higher extent of alkylation by MNU and a slower rate of repair by AGT for sites in which a guanine or modified guanine is preceded by a purine rather than a pyrimidine may explain, at least in part, mutational hot spots.

Base Sequence↗
PubMed1987

Specificity of O6-alkylguanine-DNA alkyltransferase.

Extensive investigations of the specificity of O6-alkylguanine-DNA alkyltransferase (AAT) have been carried out. These studies have shown that: (i) the mammalian protein differs from that of Escherichia coli in lacking the ability to remove methyl groups from O4-methylthymine; (ii) the protein can remove longer alkyl groups from the O6 position but the rate of repair declines as the chain length increases; (iii) O6-methylguanine in RNA is much less active as a substrate for the protein than O6-methylguanine in double-stranded DNA; (iv) the free-base O6-alkylguanine is a very weak substrate for the protein so that reaction with it leads to the loss of alkyltransferase activity. (This property can be used to deplete AAT in cultured cells and in tissues and tumours after administration of O6-methylguanine); and (v) oligodeoxynucleotides containing O6-methylguanine are substrates for AAT. Such oligodeoxynucleotides can be labelled with 32P at very high specific activity and can be used in an ultrasensitive assay for AAT activity.

Alkylation↗
PubMed1986

Effect of O6-alkylguanine pretreatment on the sensitivity of human colon tumor cells to the cytotoxic effects of chloroethylating agents.

Two human colon carcinoma cell lines which differ greatly in their content of O6-alkylguanine-DNA alkyltransferase were analyzed for their response to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 2-chloroethyl(methylsulfonyl)methanesulfonate (ClEtSoSo) before and after treatment with O6-alkylguanines. HT29 cells contained about 17 times more alkyltransferase activity than BE cells. The alkyltransferase activity of HT29 cells was reduced by 60-80% by treatment for 24 h with 0.05-0.4 mM O6-methylguanine or O6-n-butylguanine. Such pretreatment prior to exposure to CCNU or ClEtSoSo increased the sensitivity of HT29 cells to these drugs. The exposure to O6-alkylguanines gave a greater enhancement of the toxic effects of ClEtSoSo than of CCNU. There was no significant increase in the toxicity of these agents towards the BE cells which contained much lower levels of the alkyltransferase. When added alone neither O6-methylguanine nor O6-n-butylguanine showed any toxicity towards HT29 or BE cells at the doses used. These results provide strong evidence that the formation of adducts at the O6-position of guanine by these agents contributes significantly to their lethality and that this reaction is more critical for ClEtSoSo than CCNU. The enhancement of the activity of chloroethylating agents by pretreatment with nontoxic doses of O6-alkylguanines may be clinically useful in terms of increasing their therapeutic efficacy towards cells containing high levels of alkyltransferase.

Carcinoma↗
PubMed1985

Exposure of HeLa cells to 0(6)-alkylguanines increases sensitivity to the cytotoxic effects of alkylating agents.

Exposure of HeLa S3 cells to 0.4mM 0(6)-methylguanine or 0(6)-n-butylguanine for 24 h led to a substantial decrease in the activity of 0(6)-alkylguanine-DNA alkyltransferase. Such pretreatment caused a marked increase in the sensitivity of the cells to the cytotoxic effects of the cross-linking alkylating agent 1-(2-chloroethyl)-1-nitroso-3-cyclohexylurea and a smaller increase in the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. These results indicate that the repair of DNA by the alkyltransferase plays an important role in the protection of cells from the cytotoxic effects of certain alkylating agents particularly those such as 1-(2-chloroethyl)-1-nitroso-3-cyclohexylurea which ultimately lead to the formation of lethal interstrand cross-links.

Cell Division↗
PubMed1985

Extent of formation of O4-methylthymidine in calf thymus DNA methylated by N-methyl-N-nitrosourea and lack of repair of this product by rat liver O6-alkylguanine-DNA-alkyltransferase.

Calf thymus DNA was methylated by reaction with N-[3H]-methyl-N-nitrosourea and the content of O6-methyldeoxyguanosine, 3-methylthymidine and O4-methylthymidine was determined. It was found that O4-methylthymidine represented only 0.06 +/- 0.02% of the total methylation and that the ratio of O6-methyldeoxyguanosine:O4-methylthymidine was 126 +/- 31. 3-Methylthymidine represented only 0.05 +/- 0.01% of the total radioactivity and the ratio of O6-methyldeoxyguanosine:3-methylthymidine was 171 +/- 16. The ability of O6-alkylguanine-DNA-alkyltransferases from Escherichia coli and from rat liver to repair O4-methylthymidine was determined using this methylated DNA as a substrate. When the methylated DNA substrate was incubated with an excess of either of the O6-alkylguanine-DNA-alkyltransferases greater than 95% of the O6-methyldeoxyguanosine was removed. The E. coli O6-alkylguanine-DNA-alkyltransferase also removed 89% of the O4-methylthymidine but the rat liver alkyltransferase did not alter the content of O4-methylthymidine. These results indicate that the mammalian O6-alkylguanine-DNA-alkyltransferase is specific for O6-methylguanine and differs from the bacterial protein in that it does not demethylate O4-methylthymine at any significant rate. This shows that the rat O6-alkylguanine-DNA-alkyltransferase is not able to protect against the possible hazards of the promutagenic lesion, O4-methylthymidine, but the very low extent of formation of this product may limit its significance in carcinogenesis and mutagenesis.

Animals↗