DNA glycosylase enzymes induced during chemical adaptation of M. luteus.

Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively.     

Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.     

The incorporation of O6-methyldeoxyguanosine and O4-methyldeoxythymidine monophosphates into DNA by DNA polymerases I and alpha

The modified nucleoside 5'-triphosphates O6-MedGTP ad O4-MedTTP have been synthesised and their acceptability as DNA-precursors investigated using DNA polymerases I and alpha in an in vitro assay. O6-MedGMP is only incorporated into newly-synthesized DNA-like material in the presence of templates containing thymine bases. Similarly O4-MedTMP is only incorporated in the presence of templates containing guanine bases. The results confirm the promutagenic nature and base-pairing properties of O6-MeG ad O4-MeT.     

Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.     

Purification of the human O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase, the latter to apparent homogeneity

Uracil-DNA glycosylase, the enzyme that catalyzes the release of free uracil from single-stranded and double-stranded DNA, has been purified 26,600-fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil-DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme. This report also presents a 700-fold purification of HeLa S3 cell O6-methylguanine-DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.     

Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

The technique of site-directed mutagenesis has been used to investigate the mutagenicity of O6-methylguanine (O6-MeG) or hypoxanthine introduced as a single lesion at a specific locus in an M13mp9 RF molecule constructed in vitro. Following transformation of O6-MeG-containing RF molecules into E. coli JM101, mutant progeny phage were produced at a frequency not significantly different from that observed with wild-type M13mp9 RF. The mutant yield was greatly enhanced by exhausting cellular O6-MeG DNA-methyltransferase before transformation. In contrast, hypoxanthine exhibited miscoding mutagenesis in the absence of interference with cellular repair mechanisms. This indicates that cellular hypoxanthine-DNA glycosylase acts inefficiently in the removal of hypoxanthine from DNA in vivo. The precise mutational changes induced by hypoxanthine were determined by DNA sequence analysis.     

Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli

The three miscoding alkylated pyrimidines O2-methylcytosine, O2-methylthymine and O4-methylthymine are specifically recognized by Escherichia coli DNA repair enzymes. The activities are induced as part of the adaptive response to alkylating agents. O2-Methylcytosine and O2-methylthymine are removed by a DNA glycosylase, the alkA+ gene product, which also acts on N3-methylated purines. O4-Methylthymine is repaired by a methyltransferase, previously known to correct O6-methylguanine by transfer of the methyl group to one of its own cysteine residues. It is proposed that certain common structural features of the various methylated bases allow each of the two inducible repair enzymes to recognize and remove several different kinds of lesions from alkylated DNA.     

Repair of alkylated DNA: Drosophila have DNA methyltransferases but not DNA glycosylases

DNA methyltransferase activity has been identified in crude extracts of Drosophila melanogaster pupae for the removal of methyl groups from O-6 methylguanine appearing in alkylated DNA. Additionally, N-7 methylguanine and 3 methyladenine appear to be uniquely susceptible to methyltransferase activity that resides in Drosophila pupae. Consistent with this, tests to detect DNA glycosylase activity for the repair of the latter two modified bases was unsuccessful, even though a substantial loss of methyl groups from these bases was observed. Conversely, the repair of methylated purines was not detected in extracts of Drosophila embryos. The removal of methyl groups from methylated purines was dependent upon incubation temperature and was proportional to the amount of protein added to reaction mixtures. Results indicate that the methyl group is attached to protein during the repair of methylated DNA, suggesting that it is similar to the O6-methylguanine-DNA methyltransferase identified in other organisms. Although other explanations are possible, the inability to detect DNA glycosylase activity suggests that Drosophila may not rely on base excision repair for the removal of modified or nonconventional basis in DNA.     

Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase

An inducible methyltransferase of Escherichia coli acts on O6-methylguanine in DNA by conveying the methyl group to one of its own cysteine residues. The protein has now been purified to apparent homogeneity from a constitutively expressing strain. The homogeneous methyltransferase exhibits no DNA glycosylase or endonuclease activity on alkylated DNA. Further, the methyltransferase activity is strikingly resistant to heat inactivation under reducing conditions. The protein has Mr = 18,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient and Stokes radius of the native enzyme yield Mr = 18,400. The amino acid composition of the purified protein shows 4 to 5 cysteine residues/transferase molecule. The methylated, inactive form of the transferase has an unaltered molecular weight.     

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.     

Similar repair of O6-methylguanine in normal and ataxia-telangiectasia fibroblast strains. Deficient repair capacity of lymphoblastoid cell lines does not reflect a genetic polymorphism

The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.     

Enzymatic repair of premutagenic DNA lesions in human epidermis. Quantitation of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities

Extracts of human epidermis prepared by the suction blister method were used to measure O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities. Although both activities were detected in all extracts examined, a 4-5-fold interindividual variation in activity was found. No obvious correlation of the two enzyme activities with the age of the patient was observed. Neither was there any correlation between the level of uracil-DNA glycosylase activity and O6-methylguanine-DNA methyltransferase activity.     

Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase has been used as a reagent to analyse the initial reaction sites of alkylating agents such as chloroethylnitrosourea that cross-link DNA. The transferase can be employed for this purpose because it removes substituted ethyl groups from DNA, as shown by its ability to act on O6-hydroxyethylguanine residues in DNA. The enzyme counteracts the formation of interstrand cross-links induced by bis-chloroethylnitrosourea, but not those induced by nitrogen mustard. Once formed, chloroethylnitrosourea-induced cross-links are not broken by the enzyme. In agreement with deductions from experiments with living cells, it is concluded that chloroethylnitrosourea act by forming reactive monoadducts at the O6 position of guanine and/or the O4 position of thymine, which subsequently generate -CH2CH2- bridges to the complementary DNA strand. A new method for quantitating interstrand cross-links in DNA has been employed.     

Repair of alkylation damage in the fungus Aspergillus nidulans

The repair of alkylation damage in Aspergillus nidulans was investigated. We have assayed soluble protein fractions for enzymes known to be involved in the repair of this type of damage in DNA. The presence of a glycosylase activity that can remove 3-methyladenine from DNA was demonstrated, as well as a DNA methyltransferase activity that appears to act against O6-methylguanine. In addition to this approach, a series of mutants were isolated which display increased sensitivity to alkylating agents (sag mutants). 5 such mutants were further characterized, and at least 4 are shown to map to genes which have not previously been characterized. The behaviour of double mutant combinations demonstrates the existence of at least 2 pathways for the repair of alkylation damage. The majority of the sag mutants (sagA1, sagB2, sag4 and sagE5) exhibit an increased sensitivity to a range of alkylating agents, but not to UV light, while sagC3, when irradiated at the germling stage, also shows sensitivity to UV. None of the mutants isolated are defective in either the 3-methyladenine DNA glycosylase activity, or the DNA methyltransferase activity, and the nature of the defects in these strains remains to be determined.     

Comparative analysis of O6-methylguanine methyltransferase activity and cellular sensitivity to alkylating agents in cell strains derived from a variety of animal species

Using 26 cultured cell lines derived from 17 different animal species, we have measured both the activity of O6-methylguanine (O6-MeG) methyltransferase (MT) in cell extracts and the sensitivity of the strains to the lethal effects of the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). The MT activity was assayed by measuring the amount of 3H radioactivity transferred from methyl-[3H]-labeled O6-MeG in DNA to acceptor protein molecules in the extracts. In all the 21 mammalian cell strains, lethal sensitivity to ACNU as measured by colony-forming ability correlated well with cellular MT activity, indicating that the major lethal ACNU damage is reparable by the MT. On the other hand, MNNG sensitivity did not necessarily correlate with the MT activity.     

Detection of glycosylase, endonuclease and methyltransferase enzyme activities using immobilized oligonucleotides

A novel rapid assay for detection of DNA glycosylase, restriction endonuclease, and DNA methyltransferase enzyme activities is presented. The assay is based on enzyme-dependent label release (in case of glycosylase and endonuclease), or non-release (in case of methyltransferase) into solution from end-labeled DNA immobilized on solid support (CPG or Tenta Gel S-NH2). The assay has been validated for monitoring activity of repair enzyme uracil-DNA glycosylase, restriction endonucleases SsoII, MvaI and EcoRII and (cytosine-5)-DNA methyltransferase SsoII. Two types of labels have been tested and found compatible with the assay: radioactive (32P) and fluorescent (rhodamine B and fluorescein). The enzyme activity is estimated as a ratio of the label released into solution to the total amount of the label. Use of fluorescent labeling facilitates detection while use of solid phase-immobilized substrates facilitates product separation, improved assay sensitivity, and increases throughput of assay. Proposed technique provides an estimate of enzyme activity but not its specific activity. Thus, the assay will most valuable in the applications where rapid estimation of enzyme activity is necessary.     

Repair of O6-ethylguanine DNA lesions in isolated cell nuclei. Presence of the activity in the chromatin proteins

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6-ethylguanine and 7-ethylguanine during a subsequent incubation at 37 degrees C. The rate of O6-ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6-ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7-ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7-ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase. The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6-ethylguanine from an added ethylated DNA. No free O6-ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6-ethylguanine were released either, indicating that the disappearance of O6-ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease. Activities capable of removing O6-ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2-3-times the repair activity of the chromatin proteins.     

Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage.

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.