Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E. coli

DNA substrates containing O⁶-n-butylguanine, O⁶-iso-butylguanine,O⁶-n-propylguanine and O⁶-iso-propylguanine were prepared byreaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea.These substrates were used to test the ability of O⁶-alkylguanine-DNAalkyltransferases from Escherichia coli and rat liver to removesuch alkyl groups from the O⁶-position of guanine. It was foundthat all of these adducts were removed by the alkyltransferases,but the branched alkyl chain iso-butyl- and iso-propyl adductswere removed very slowly. Also, when tested with a DNA substratecontaining both O⁶-n-propylguanine and O⁶-iso-propylguanine,the alkyltransferases removed almost all of the n-propyl-adductbefore the iso-propyl-adduct was attacked. Both alkyltransferasesshowed a decreasing rate of reaction as the size of the alkylgroup increased, but there was a significant difference betweenthe rat liver and E. coli alkyltransferase in the relative rates.The rat liver alkyltransferase repaired O⁶-methylguanine moreslowly than the E. coli protein, but was considerably more rapidthan the bacterial equivalent when acting on n-propyl- and n-butyl-adducts.The relative rates of repair were methyl > ethyl > n-propyl> n-butyl >iso-propyl, iso-butyl for the E. coli alkyltransferaseand methyl > ethyl, n-propyl > n-butyl > iso-propyl,iso-butyl > 2-hydroxyethyl for the rat liver protein. Theseresults indicate that differential rates of repair may contributeto the relative risks of carcinogenesis and mutagenesis by exposureto alkylating agents of different size and that rates of repairmay be species specific and must be determined from specificmeasurements rather than extrapolated from data on other organisms.     

Differential effects of procarbazine and methylnitrosourea on the accumulation of O6-methylguanine and the depletion and recovery of O6-alkylguanine-DNA alkyltransferase in rat tissues

The kinetics of accumulation of the premutagenic DNA adductO⁶-methylguanine (O⁶-meG) in the liver, blood leukocytes, lymphnodes and bone marrow of rats was examined and compared aftersingle or multiple doses of procarbazine, a methylating cytostaticdrug employed in the treatment of Hodgkin's lymphoma patients,and methylnitrosourea (MNU), an experimental methylating agentand carcinogen. Maximal O⁶-meG levels occurred 1–2 h afteradministration of single doses of procarbazine (10 mg/kg) orMNU (1 mg/kg), thereafter decreasing with halflives of     

Comparison of the rates of repair of O6-alkylguanines in DNA by rat liver and bacterial O6-alkylguanine-DNA alkyltransferase

The rates of loss of O6-methylguanine and O6-ethylguanine from rat liver DNA were determined over a time period of 15 min to 4 hr after various doses (5 micrograms/kg to 2 mg/kg) of dimethylnitrosamine and diethylnitrosamine which produced total amounts of these adducts in the range of 300 to 16,000 molecules/cell. This amount is considerably less than the content of O6-alkylguanine-DNA alkyltransferase protein (approximately 60,000 molecules/hepatocyte), and during the time period studied, the adducts were found to be lost with pseudo-first order kinetics. The half-life for O6-methylguanine was 47 min. O6-Ethylguanine was removed 3.6 times more slowly with a half-life of 172 min. The ability of partially purified rat liver O6-alkylguanine-DNA alkyltransferase to remove O6-methylguanine and O6-ethylguanine from [3H]alkyl-labeled DNA substrates in vitro was measured, and it was found that O6-methylguanine was removed 3.4 times more rapidly than was O6-ethylguanine. These results are consistent with the hypothesis that most, if not all, of the repair of these adducts which occurs within the first 4 hr after treatment is due to the alkyltransferase protein. Diethylnitrosamine, which is slightly more potent as a carcinogen to rat liver, produced a total amount of O6-ethylguanine of 3.7 mumol/mol guanine/mg compared to O6-methylguanine (28 mumol/mol guanine/mg) given by dimethylnitrosamine. The slower rate of loss of the ethyl adduct is not sufficient to account for this difference, and the results, therefore, support the concept that other DNA adducts (possibly O-alkylpyrimidines) contribute to the initiation of tumors by diethylnitrosamine. Preliminary evidence that the rat liver alkyltransferase can also remove hydroxyethyl groups from DNA at a rate slower than removal of ethyl groups was also obtained. Bacterial O6-alkylguanine-DNA alkyltransferase was shown to remove methyl, ethyl, and hydroxyethyl groups from the O6 position of guanine in DNA using fluorescence detection to quantitate these adducts. The bacterial protein removed methyl groups very rapidly but was much slower than the rat liver protein on the larger adducts. These results suggest that the relative rates of repair of different alkyl groups may be species specific and must be determined experimentally in the cell of interest before conclusions concerning biological effects can be drawn.     

Comparative dosimetry of O6-methylguanine in humans and rodents treated with procarbazine

The accumulation of O⁶-methylguanine (O⁶-meG) in the DNA ofblood leukocytes of 21 Hodgkin's lymphoma patients (followedfor up to 12 cycles of treatment) treated in the context ofMOPP combination chemotherapy with 150 mg procarbazine dailyfor 10 days was examined and compared to that observed in ratstreated with different doses of procarbazine as a single agentonce per day for 10 days. In humans, the adduct accumulatedin a doserelated fashion and appeared to approach a steady-stateafter 7–8 days of treatment Adduct levels on day 10 ofthe treatment cycle averaged 0.25 ± 0.09 (mean ±SD) µmol/molG and, for different individuals, covereda 3-fold range. Intra-individual variability between differenttreatment cycles was much more limited than inter-individualvariability, the two parameters accounting for 8.9% and 84.5%respectively of adduct variance at a constant cumulative dose.Comparison of the dose-response relationships for humans andrats indicates that, under conditions of no depletion of O⁶-alkylguanine-DNAalkyltransferase (AGT), O⁶-meG accumulates in blood leukocyteDNA of humans at a rate which is only approximately 2-fold lowerthan in rats, implying that, to the extent to which O⁶-meG contributesto the genotoxic activity of procarbazine, human susceptibilityto it is likely to be comparable to that of the rat This islikely to be true also of the bone marrow (the tissue of interestas a target tissue for leukaemogenesis), since the tissue distributionof O⁶-meG induced by low doses of procarbazine in rats, miceand rabbits indicated that blood leukocyte levels of this adductclosely reflect those in the bone marrow. Based on these results,it is estimated that by the end of a MOPP chemotherapy cycleO⁶-meG reaches levels of the order of 0.2–0 3 fmol/µgDNA (03–0.5 µmol/molG) in human bone marrow (thetarget tissue of leukaemogenesis observed after such treatment).     

Comparison of O6-alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues

O⁶-Alkylguanine-DNA alkyltransferase (alkyltransferase) is therepair protein for O⁶-alkylguanine, a pre-mutagenic adduct formedby a variety of alkylating agents. Previous comparisons of therepair capacity of O⁶-alkylguanine in different tissues haveexpressed the alkyltransferase activity relative to total protein,and have asserted that tissues with low levels of activity wereat greater risk for mutagenic damage than tissues with higherlevels of activity. Because the alkyltransferase uses DNA assubstrate, and because tissues vary greatly in protein content,comparisons of tissue alkyltransferase activity may be moreappropriately based on cellular DNA content. We compared alkyltransferaseactivity relative to tissue DNA content with the activity relatedto protein content in human, rat and mouse tissues. In eachspecies, liver containing the highest level of activity usingeither method. In agreement with the findings of others, lowlevels of alkyltransferase activity relative to protein wereseen in human brain, rat brain and small intestine, and mousekidney. However, based on alkyltransferase activity relativeto DNA content, low levels of activity were seen in human bonemarrow myeloid precursors, rat bone marrow, brain and intestine,and mouse spleen and bone marrow. The range of activity betweentissues was 18-fold in human, 15-fold in rat and 8-fold in mouse.In general, the rank of alkyltransferase activity relative toDNA for each tissue was human > rat > mouse. These resultssuggest that the mouse is more susceptible to nitrosoureas thanrat or human. In each species, the organs with low levels ofalkyltransferase activity relative to tissue DNA content wouldappear to be targets for mutagenic damage following nitrosoureaexposure.     

Activity of O6-alkylguanine-DNA alkyl transferase in the liver, kidney and white blood cells of rats of different ages.

Age-related differences in the sensitivity of rats to alkylating carcinogens may be dependent on various factors, including the cellular levels of O6-alkylguanine-DNA alkyltransferase (AT). In the present study, the levels of AT were measured in protein extracts prepared from liver, kidney and peripheral white blood cells of male outbred rats aged 1, 4, 14, 22 and 36 months. The AT level (expressed as activity per milligram protein) in liver extracts was lower in rats aged 1, 4 or 36 months than in extracts prepared from rats aged 14 or 22 months. This observation of a variation in AT level with age is in agreement with our previous results. The AT levels in kidney and white blood cells did not differ significantly with age, and in all cases the AT levels were lower than those observed in the liver extracts, the kidney extracts having more AT activity than the white blood cell extracts. The total protein content of both liver and kidney tissues, calculated per gram of wet tissue, increased to a maximum at 14 months and subsequently declined, the total protein content being always higher in the liver than in the kidney. In contrast, the DNA content per gram of wet tissue was highest in young animals and subsequently declined to a minimum at 14 months. The implications of this inverse relationship to the levels of AT activity are discussed.     

O6-alkylguanine-DNA alkyltransferase activity in human brain tumors

The O6-alkylguanine-DNA alkyltransferase (AT) activity in different kinds of human brain tumors was investigated. Twenty-seven brain tumors were analysed. Twenty-five of them showed proficient AT activity with values ranging between 20 and 722 fmol AT/mg protein. The two AT-deficient tumors observed were an oligodendroglioma and an astrocytoma. The relationship between the different histological kinds of tumor, with respect to the AT activity was: meningeomas greater than sarcomas greater than glioblastomas greater than astrocytomas greater than oligodendrogliomas greater than neurinomas greater than lymphomas. The proposal of Kohn (DNA filter elution methods in anticancer drug development. In: Concepts, Clinical Developments, and Therapeutic Advances in Cancer Chemotherapy. Editor: F.M. Muggia. Martinus Nijhoff Publishers, Boston) to confine treatments with alkylating antineoplastic agents to AT-deficient tumors, is discussed.     

O6-alkylguanine-DNA alkyltransferase activity in normal human tissues and cells

Normal adult human tissues and cultured bronchial epithelial cells and fibroblasts exhibit O6-alkylguanine-DNA alkyltransferase activity in vitro by catalyzing the repair of the promutagenic alkylation lesion O6-methylguanine from DNA. The amount repaired by extracts of liver, peripheral lung, and colon extracts was proportional to the amount of extract protein. Repair of O6-methylguanine led to stoichiometric regeneration of guanine in the DNA and stoichiometric formation of S-methylcysteine in protein. Alkyltransferase activity varies in the different human tissues tested in the decreasing order of liver greater than colon greater than esophagus greater than peripheral lung greater than brain. Extracts of lung tissues, cultured human bronchial epithelial cells, and fibroblasts had similar alkyltransferase activities. Various human tissues exhibit 2- to 10-fold higher alkyltransferase activity than corresponding rat tissues. Whereas the interindividual variation of the activity was 4- to 5-fold in ten or more human lung and colon specimens, the interindividual variation in the inbred rat was less than 20%. The present results show that different human tissues and cells have a several-fold higher capacity to repair O6-methylguanine in DNA than do rat tissues and that the repair process occurs via a mechanism similar to that shown previously in other mammalian cells and Escherichia coli.     

Interferon inducers increase O6-alkylguanine-DNA alkyltransferase in the rat liver

We investigated whether treatment with the interferon inducer polyinosinic-polycytidylic acid and other cytokines (interleukin-1, tumor necrosis factor) or the cytokine inducer lipopolysaccharide modified O6-alkylguanine-DNA alkyltransferase (AT) in rat liver. AT levels were determined in liver extracts using N-[3H]methyl-N-nitrosourea alkylated calf thymus DNA as substrate and an HPLC procedure to measure O6-methylguanine. Doses as low as 0.1 mg/kg i.p. of polyinosinic-polycytidylic acid caused a highly significant increase (P less than 0.01) in AT levels in the liver, evident either 24 or 48 h after treatment. Lipopolysaccharide at the dose of 80 micrograms/kg i.p. also induced AT whereas interleukin-1 (60 micrograms/kg) or tumor necrosis factor (60 micrograms/kg) were inactive. Treatment with human recombinant interferon alpha A/D caused a highly significant increase in AT levels, thus confirming the hypothesis that interferon was probably responsible for the observed effect. These results suggest a link between the immune response and DNA repair mechanisms.     

The role of tyrosine-158 in O6-alkylguanine-DNA alkyltransferase activity

The tyrosine residue present at position 158 in the human O⁶-alkylguanine-DNAalkyltransferase is one of 22 amino acid residues that are conservedin all known alkyltransferase protein sequences. The importanceof this amino acid in the reactions brought about by the alkyltransferasewas studied by changing this residue to alanine or to phenylalanine.The control and mutant alkyltransferase proteins were expressedin an Escherichia coli strain lacking alkyltransferase activityand the proteins purified to near homogeneity and their activitiesmeasured using both methylated DNA and O⁶-benzylguanine (BG)as substrates. The alteration to alanine led to a very largedecrease in activity towards both substrates but removal ofO⁶-methylguanine from DNA and the conversion of BG to guaninecould still be detected when large amounts of the protein wereused. The activity of the Y158A mutant was at least 800 timesless than that of the control alkyltransferase. The change oftyrosine-158 to phenylalanine reduced the rate of reaction withmethylated DNA only slightly (to about one-third). The conversionof BG to guanine by the Y158F mutant was also reduced to aboutone-third when assayed in the absence of DNA and by about one-halfin the presence of DNA. These results suggest that the presenceof tyrosine at position 158 plays an important but not absolutelyessential role in the reactions brought about by the alkyltransferase.This role is likely to involve the stabilization of the boundsubstrate by interaction with the aromatic ring of the tyrosine.The hydrogen bond formed by the hydroxyl group from tyrosine-158may also facilitate the reaction but the contribution from thisinteraction is relatively small.     

Gamma glutamyl transferase activity in rat colon during experimental colonic carcinogenesis

Colonic gamma glutamyl transferase activity is raised in colonic malignancy. This study was undertaken to determine how early and how extensively this raised activity occurs during carcinogenesis. Sixty rats were given weekly injections of dimethylhydrazine for 16 weeks, and 20 rats acted as controls. Groups of treated and control rats were sacrificed at regular intervals during the pre-cancerous phase of carcinogenesis, and gamma glutamyl transferase activity was measured at several sites in the colon. Gamma glutamyl transferase activity was raised as early as 4 weeks after the first injection of dimethylhydrazine (p less than 0.05), and the elevation persisted until the development of overt tumours. The raised gamma glutamyl transferase activity was observed throughout both the proximal and distal colon long before the appearance of macroscopic tumours. We conclude that an early and widespread elevation of colonic gamma glutamyl transferase activity occurs during carcinogenesis. This may have useful applications in the early detection of colonic malignancy.     

Smoking-related increase in O6-alkylguanine-DNA alkyltransferase activity in human lung tissue

O⁶-Alkylguanine-DNA alkyltransferase (AGAT) activity was determinedin macroscopically normal peripheral lung tissues from 122 patientsundergoing lung surgery. AGAT activity of smokers was 1.4-foldhigher than that of lifetime non-smokers (P = 0.030). Less thanone year of abstinence from smoking did not cause a significantdecrease in AGAT activity in former smokers, however, longerabstinence resulted in a decrease in AGAT activity to the leveldetected in lifetime non-smokers. There was no significant differencebetween levels of AGAT activity in lung cancer and noncancerpatients. The results demonstrate that the level of AGAT activityin human peripheral lung tissue is influenced by smoking habitsbut does not have a diagnostic correlation to lung cancer.     

Inactivation of purified human O6-alkylguanine-DNA alkyltransferase by alkylating agents or alkylated DNA

O6-Alkylguanine-DNA alkyltransferase partially purified from cultured human lymphoblasts (CEM-CCRF line) was inactivated with DNA substrates that had been treated separately with five methylating agents or with five chloroethylnitrosoureas. The extent of depletion of the transferase by alkylated DNA was compared with its inactivation by direct reaction with these ten agents. As expected, DNA substrates treated with methylating agents that efficiently produce O6-methylguanine were most effective in depleting the transferase, as was DNA pretreated with 1-(2-chloroethyl)-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and chlorozotocin, agents presumed to form O6-chloroethylguanine as well as O6-hydroxyethylguanine in DNA. Unexpectedly, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-trans-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea were relatively ineffective in producing a DNA substrate that would inactivate the transferase, suggesting that both agents produce low steady-state levels of O6-alkylguanine. All of the agents tested were capable of inactivating the transferase by direct alkylation, although the efficiency of this activity ranges widely. We conclude that simple methylating agents and the cross-linking chloroethylnitrosoureas can inactivate O6-alkylguanine-DNA alkyltransferase both directly and indirectly, affording two mechanisms by which such agents could modulate their own cytotoxicity.     

Inactivation of O6-alkylguanine-DNA alkyltransferase in the rat liver due to an intraperitoneal administration of methylated DNA

Intraperitoneal administration of 6.5 mg of in vitro methylatedDNA (meDNA) containing 1.5 x 10^–44 mM of O⁶-methylguanine(6MG) to male outbred rats weighing 150 g led to a considerabledecrease in the activity of liver O⁶-alkylguanine - DNA alkyltransferase(AT). One hour after treatment there occurred a 4- to 5-folddecrease in the AT activity followed by its slow recovery. However,after 48 h, AT activity considerably exceeded control levels.A 5-fold decrease in the amount of administered meDNA resultedin the absence of its effect, whereas administration of higheramounts produced a further AT inactivation. A similar treatmentwith non-methylated DNA did not change AT activity. The possibilityof AT exhaustion under in vivo conditions and thereby inhibitionof repair of O⁶-alkylguanine in DNA, playing a key role in mutagenic,carcinogenic and antineoplastic effects of certain alkylatingagents, might be helpful in increasing susceptibility of animalsto such compounds.     

Inactivation of O 6-alkylguanine-DNA alkyltransferase by 8-substituted O 6-benzylguanine analogs in mice

PURPOSE: The purpose of this study was to determine the usefulness of various 8-substituted O6-benzylguanine (BG) analogs as modulators of the DNA repair protein. O6-alkylguanine-DNA alkyltransferase (AGT). More specifically, the degree of inactivation of AGT in mouse brain, liver, kidney and tumor by O6-benzyl-8-oxoguanine (8-oxoBG), 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-bromoBG) and O6-benzyl-8-trifluoromethylguanine (8-tfmBG) was compared to inactivation by BG, a modulator in phase II clinical trials. BG is converted rapidly to 8-oxoBG in rodents, monkeys and humans. It was reasoned that 8-substituted analogs of BG would exhibit different pharmacological properties compared to BG which could influence tissue bioavailability and, thus, the extent of AGT inactivation in vivo. We compared the tissue distribution of these agents and AGT activity following administration of the 8-substituted analogs. MATERIALS AND METHODS: At various time points up to 24 h after i.p. administration of the BG analogs, tissues (i.e. brain, liver, kidney), A549 lung tumor xenografts (i.p.) or D456 brain tumor xenografts (i.c.) were harvested from athymic nude mice for AGT analysis. AGT activity was quantified in tissue extracts using a biochemical assay with [3H]methylated DNA as a substrate. In addition, concentrations of BG and 8-oxoBG were determined by HPLC with fluorescence detection in mouse tissues following administration of drug. RESULTS: Each of the 8-substituted analogs of BG demonstrated variable AGT inactivation capabilities that were comparable to or better than those of BG especially in kidney and brain tissues. There was a more pronounced depletion of AGT inactivation in brain and D456 brain tumor xenografts following administration of BG compared to 8-oxoBG that could be explained by a much greater concentration of AGT-inactivating drug (BG plus the metabolite 8-oxoBG for mice treated with BG versus 8-oxoBG for mice treated with 8-oxoBG) present in these tissues. The AUCs for brain, kidney and liver were 3.2, 6.9 and 1 1.8 times greater for BG than for 8-oxoBG. CONCLUSIONS: 8-substituted analogs of BG possess unique AGT-inactivation profiles in vivo that are different from that of BG. The AGT-inhibitory activities of BG and its major metabolite, 8-oxoBG, are related to tissue disposition of both drugs.     

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

Summary 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.Abbreviations AGT O⁶-alkylguanine-DNA alkyltransferase - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - Clomesone 2-chloroethyl(methylsulfonyl) methanesulfonate - HPLC high-pressure liquid chromatography     

Measurement of O6-alkylguanine-DNA alkyltransferase activity in human cells and tumor tissues by restriction endonuclease inhibition

A sensitive assay for O6-alkylguanine-DNA alkyltransferase activity in cell or tumor extracts has been devised. The theoretical basis of the new assay lies in the observation that certain restriction enzymes will not cleave DNA containing methylated bases. Thus, if a synthetic oligodeoxynucleotide with a restriction sequence containing O6-methylguanine were incubated with the restriction enzyme, this synthetic oligodeoxynucleotide should remain intact. However, if the guanine-O6 methyl group were first removed by O6-alkylguanine-DNA alkyltransferase present in certain cell or tissue extracts the synthetic oligodeoxynucleotide would be cleaved by the restriction enzyme. The parental oligodeoxynucleotide and its restriction products are separated from each other and analyzed on denaturing polyacrylamide gels. The extent of cleavage by the restriction enzyme is a direct assay of the content of O6-alkylguanine-DNA alkyltransferase in the cell/tumor extracts. The assay has been tested against cell culture and xenograft tumor systems and has performed in a predictive manner, correctly predicting five Mer- and three Mer+ phenotypes. Furthermore, the assay is quantitative and the number of molecules of the O6-alkylguanine-DNA alkyltransferase per cell estimated using this assay agrees with those that have been published.     

Inactivation of O6-alkylguanine-DNA alkyltransferase in HeLa cells by cisplatin

Inactivation of O⁶-alkylguanine-DNA alkyltransferase (O⁶-AGT)in HeLa CCL₂ cells by cisplatin was studied. HeLa CCL₂ cellstreated with cisplatin showed a dose-dependent decline in O⁶-AGTactivity. After cisplatin was removed and replaced with freshmedium, the transferase level began to rise slowly. By 72 hslightly more than 80% of the activity was recovered. It seemsthat the activity of the alkyltransferase can be inactivatedby platinated DNA adducts. The data suggest that the O⁶-platinum-guanineformation and the O⁶-alkyltransferase depletion are not responsiblefor cytotoxicity but may result in a base substitution mutationin mammalian cells.     

Repair of synthetic oligonucleotides containing O6-methylguanine, O6-ethylguanine and O4-methylthymine, by O6-alkylguanine-DNA alkyltransferase

32P-Labelled self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, and O4-methylthymine have been synthesized and used as substrates for the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AAT). The reaction was second-order with rate constants of 2.6 X 10(7) M-1 sec-1 for O6-methylguanine, 2.6 X 10(4) M-1 sec-1 for O6-ethylguanine and 2.5 X 10(3) M-1 sec-1 for O4-methylthymine. These oligomers should allow sensitive and specific assay of the mammalian enzyme.