Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli

The qualitative and quantitative features of mutagenesis by two DNA adducts of carcinogenic alkylating agents, O6-methylguanine (m6G) and O4-methylthymine (m4T), were examined in vivo. The deoxyhexanucleotides 5'-GCTAGC-3' and 5'-GCTAGC-3' were synthesized, where the underlined bases are the positions of m4T or m6G, respectively. By use of recombinant DNA techniques, the respective hexanucleotides or an unmodified control were inserted into a six-base gap in the otherwise duplex genome of the Escherichia coli virus M13mp19-NheI. The duplex adducted genome was converted to single-stranded form and introduced into an E. coli strain that was phenotypically normal with regard to m6G/m4T repair, a strain deficient in repair by virtue of an insertion in the gene encoding the Ada-m6G/m4T DNA methyltransferase, or the same two cell lines after challenge with N-methyl-N'-nitro-N-nitrosoguanidine. Treatment with this alkylating agent chemically compromises alkyl-DNA repair functions. The mutation efficiency of m6G was low or undetectable (0-1.7%) in all cell systems tested, owing, we believe, to rapid repair. In striking contrast, the mutagenicity of m4T was high (12%) in cells fully competent to repair alkylation damage and was roughly doubled when those cells were pretreated with N-methyl-N'-nitro-N-nitrosoguanidine to suppress repair. Taken together, these data suggest that m4T is potentially more mutagenic than m6G and, if formed by a DNA methylating agent, may pose a significant threat to the genetic integrity of an organism.     

O6-methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with O6-methylguanine

Mutations were induced in the ampicillinase gene of a bacteriophage f1/pBR322 chimera both by incorporation of O6-methyl-dGTP opposite T during DNA replication in vitro and by site-directed mutagenesis using O6-methylguanine-containing oligonucleotides. After passage of the DNA through Escherichia coli, analysis of 151 O6-methyl-dGTP-induced mutations indicated a significantly greater number of unmutated mutation sites than expected, whereas the mutated sites generally fit a Poisson distribution. The unmutated sites are assumed to be caused by the inability of some sequences to tolerate the presence of a tetrahedral methyl group within the confines of a Watson-Crick helix (Toorchen, D., and Topal, M.D. (1983) Carcinogenesis 4, 1591-1597). A consensus of the DNA sequences surrounding unmutated mutation sites was derived. The consensus sequence had significant similarity to the region of the rat Harvey ras oncogene containing the N-methyl-N-nitrosourea activated site for transformation (Zarbl, H., Sukumar, S., Arthur, A. V., Dionisio, M.-Z., and Barbacid, M. (1985) Nature 315, 382-385). We propose that direct alkylation at O6 of a guanine present within the consensus sequence may produce a DNA conformation less subject to repair. Mutation by O6-methylguanine-containing oligonucleotides demonstrated that repair of the O6-methylguanine lesions varied at least 3-4-fold with position of the lesion.     

Mismatch repair proteins collaborate with methyltransferases in the repair of O(6)-methylguanine

DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O(6)-methylguanine (O(6)mG), which stably pairs with thymine during replication and thereby creates a promutagenic O(6)mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O(6)mG:T mismatches can lead to cell death or result in G:C-->A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O(6)mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O(6)mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O(6)mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O(6)mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O(6)mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O(6)mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.     

Replication past O(6)-methylguanine by yeast and human DNA polymerase eta

O(6)-Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase eta (Poleta) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in the rad30Delta pol32Delta double mutant of yeast, which lacks the RAD30 gene that encodes Poleta and the Pol32 subunit of DNA polymerase delta (Poldelta). Although Poldelta can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Poleta is much more efficient in replicating through m6G than Poldelta. Both Poleta and Poldelta insert a C or a T residue opposite from m6G; Poleta, however, is more accurate, as it inserts a C about twice as frequently as Poldelta. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Poleta could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy.     

Measurement of the removal of O6-methylguanine and O4-methylthymine from oligodeoxynucleotides using an immunoprecipitation technique

A sensitive and rapid procedure for measurement of alkyltransferase repair activity involving oligodeoxynucleotides followed by immunoprecipitation is described. Dodecadeoxynucleotides containing O6-methylguanine or O4-methylthymine were used as substrates for alkyltransferases and the reaction products of methylated or demethylated substrates were separated by precipitation with highly specific antibodies. This approach for O6-alkylguanine-DNA alkyltransferase measurement is far more rapid than when the reaction products are separated by chromatography. This technique makes the assay applicable to large-scale epidemiological or clinical studies and suggests a similar methodology could be applied for other DNA repair enzymes.     

A common mechanism for repair of O6-methylguanine and O6-ethylguanine in DNA

The mutagenic effects of several ethylating and methylating agents were assessed in Encherichia coli strains that are defective in the adaptive response to alkylating agents. These mutants were either deficient in the response or expressed it constitutively. When expressed, the repair pathway removed the major mutagenic lesion produced by either methylating or ethylating agents. This lesion was almost certainly O⁶-alkylguanine produced by alkylation of DNA, and the mechanism for its removal was characterized in vitro. E. coli cells expressing the adaptive response contain relatively large amounts of a protein that transfers the methyl group from O⁶-methylguanine to one of its own cysteine residues (Olsson & Lindahl, 1980). This methyltransferase was shown to act in an analogous fashion on O⁶-ethylguanine. Incubation of ethylated DNA with purified transferase led to disappearance of the O⁶-ethylguanine residues, and S-ethylcysteine was simultaneously generated in the protein. The greater sensitivity of E. coli wild-type to ethylating than methylating agents may be explained by a slower repair of O⁶-ethylguanine than O⁶-methylguanine and also a weaker ability of ethylating agents to induce the adaptive response.     

Specificities of human, rat and E. coli O6-methylguanine-DNA methyltransferases towards the repair of O6-methyl and O6-ethylguanine in DNA.

The behaviour of highly purified bacterial expressed rat O6-methylguanine-DNA methyltransferase (MGMT) towards the repair of CGCm6GAGCTCGCG and CGCe6GAGCTCGCG (km6G/ke6G = 1.45, where k is the second order repair rate constant determined, m6G and e6G are O6-methyl and O6-ethylguanine) is similar to that of E. coli 39kD Ada protein (km6G/ke6G = 1.6). However, the human MGMT is very different (km6G/ke6G = 163). The preferential repair of O6-ethylguanine lesion by the rat MGMT appears not to be related to the lack of the initiator methionine in the expressed protein since similar results were obtained from N-terminal Glutathione-S-transferase (GST) fused protein (GSTMGMT) which retains the methionine. The possible relationship between these findings and the differences observed in the primary amino acid sequence of these proteins is discussed. In addition the preferential repair of O6-ethylguanine substrate by the 39kD Ada protein as compared to the catalytic C-terminus alone (different by 134 times) suggests that the N-terminus plays a crucial role in the repair of O6-ethylguanine. This is in contrast to the minor effects of the GST domain when fused to the N-terminus of mammalian MGMT.     

Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins

The Escherichia coli AlkB protein repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions in DNA and RNA by oxidative demethylation, a reaction requiring ferrous iron and 2-oxoglutarate as cofactor and co-substrate, respectively. Here, we have studied the activity of AlkB proteins on 3-methylthymine (3-meT) and 1-methylguanine (1-meG), two minor lesions which are structurally analogous to 1-meA and 3-meC. AlkB as well as the human AlkB homologues, hABH2 and hABH3, were all able to demethylate 3-meT in a DNA oligonucleotide containing a single 3-meT residue. Also, 1-meG lesions introduced by chemical methylation of tRNA were efficiently removed by AlkB. Unlike 1-meA and 3-meC, nucleosides or bases corresponding to 1-meG or 3-meT did not stimulate the uncoupled, AlkB-mediated decarboxylation of 2-oxoglutarate. Our data show that 3-meT and 1-meG are repaired by AlkB, but indicate that the recognition of these substrates is different from that in the case of 1-meA and 3-meC.     

Identification and preliminary characterization of an O6-methylguanine DNA repair methyltransferase in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a DNA repair methyltransferase (MTase) that repairs O6-methylguanine. Methyl groups are irreversibly transferred from O6-methylguanine in DNA to a 25-kilodalton protein in S. cerevisiae cell extracts, and methyl transfer is accompanied by the formation of S-methylcysteine. The yeast MTase is expressed at approximately 150 molecules/cell in exponentially growing yeast cultures but is not detectable in stationary phase cells. Unlike mammalian and bacterial MTases, the yeast MTase is very temperature-sensitive, having a half-life of about 4 min at 37 degrees C, which may explain why others have failed to detect it. Like other DNA repair MTases, the S. cerevisiae MTase repairs O6-methylguanine more efficiently in double-stranded DNA than in single-stranded DNA. Synthesis of the yeast DNA MTase is apparently not inducible by sublethal exposures to alkylating agent, but rather MTase activity is depleted in cells exposed to low doses of alkylating agent. Judging from its molecular weight and substrate specificity, the yeast DNA MTase is more closely related to mammalian MTases than to Escherichia coli MTases.     

DNA-mediated transfer and expression of a human DNA repair gene that demethylates O6-methylguanine.

Human liver DNA was transfected into CHO cells (mex-) along with pSV2gpt and colonies were selected first for resistance to mycophenolic acid and then to chloroethylnitrosourea. Transformants were obtained that contained approximately 10,000 molecules of O6-alkylguanine alkyltransferase (mex+) per cell. Their genome contained at least three copies of the human Alu sequence.     

Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT)

The human DNA repair protein O⁶-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O⁶-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O⁶-alkylguanines. One such ODN containing O⁶-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC₅₀ of 0.1 nM.     

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.     

Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases

Rare DNA lesions that are chemically stable and refractory to repair may add disproportionately to the accumulation of mutations in long lived cells. 3-Methylthymine is a minor lesion that is induced by DNA-methylating agents and for which no repair process has been described previously. Here we demonstrate that this lesion can be directly demethylated in vitro by bacterial and human DNA dioxygenases. The Escherichia coli AlkB and human ABH3 proteins repaired 3-methylthymine in both single-stranded and double-stranded polydeoxynucleotides, whereas the human ABH2 protein preferred a duplex substrate. Thus, the known substrates of these enzymes now include 3-methylthymine in DNA, as well as 1-methyladenine and 3-methylcytosine, which all have structurally similar sites of alkylation. Repair of 3-methylthymine by AlkB and ABH3 was optimal at pH 6, but inefficient. At physiological pH, 3-methylthymine, which is a minor methylated lesion, was more slowly repaired than the major lesion generated in single-stranded DNA, 3-methylcytosine. Our data suggest that 3-methylthymine residues in DNA will be repaired inefficiently in vivo and therefore may occur at a low steady-state level, but the residues should not gradually accumulate to high levels in long lived cells.     

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

O⁶-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O⁶-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O⁶-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.     

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

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.     

Redesigning the substrate specificity of human O(6)-alkylguanine-DNA alkyltransferase. Mutants with enhanced repair of O(4)-methylthymine.

Human O(6)-alkylguanine-DNA alkyltransferase (MGMT) repairs potentially cytotoxic and mutagenic alkylation damage at the O(6)-position of guanine and the O(4)-position of thymine in DNA. We have used random sequence mutagenesis and functional complementation to obtain human MGMT mutants that are resistant to the MGMT inhibitor, O(6)-benzylguanine [Encell, L. P., Coates, M. M., and Loeb, L. A. (1998) Cancer Res. 58, 1013-1020]. Here we describe screening of O(6)-benzylguanine-resistant mutants for altered substrate specificity, i.e., for an increased level of utilization of O(4)-methylthymine (m(4)T) relative to that of O(6)-methylguanine (m(6)G). One mutant identified by the screen, 56-8, containing eight substitutions near the active site (C150Y, S152R, A154S, V155G, N157T, V164M, E166Q, and A170T), was purified and characterized kinetically. The second-order rate constant for repair of m(4)T by the mutant was up to 11.5-fold greater than that of WT MGMT, and the relative m(4)T specificity, k(m(4)T)/k(m(6)G), was as much as 75-fold greater. In competition experiments with both substrates present, the mutant was 277-fold more sensitive to inhibition by m(4)T than WT MGMT. This mutant, and others like it, could help elucidate the complex relationship between adduction at specific sites in DNA and the cytotoxicity and mutagenicity of alkylating agents.     

Roles of two types of O6-methylguanine-DNA methyltransferases in DNA repair

Escherichia coli possesses 2 types of O6-methylguanine-DNA methyltransferases, one inducible and the other constitutive. These enzymes are coded by the ada and the ogt genes, respectively. Using a synthetic ogt-specific probe, we mapped ogt at 29.4 min, near the 5'-flanking region of the nirR gene, on the E. coli chromosome. To elucidate the roles of the 2 types of methyltransferases in DNA repair, we constructed mutant strains which lack either one or both of the genes. In either the ada+ or the ada- background, the ogt mutation had no effect on cell survival after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. On the other hand, ada- ogt- cells were more prone to mutation as compared to the ada- ogt+ cells exposed to MNNG. The frequency of spontaneous mutation of cells defective in either one or both of the genes was the same, however, the introduction of the ogt+ plasmid into the cells produced a 2-3-fold decrease in the frequency of spontaneous mutation. O6-Methylguanine-DNA methyltransferases appear to eliminate premutagenic DNA lesions not only from cells exposed to alkylating agents but also from those grown in the absence of the agents.