Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. Suggestive evidence for O4-methylthymine repair by eukaryotic methyltransferases

The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively. This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined.     

DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.     

Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium.

Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.     

The suicidal DNA repalr methyltransferases of microbes

Virtually every organism so far tested has been found to possess an extremely efficient DNA repair mechanism to ensure that certain alkylated oxygens do not accumulate in the genome. The repair is executed by DNA methyltransferases (MTases) which repair DNA O6-methylguanine (O6MeG), O4-methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repair of just one, or sometimes two, O-alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection against death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well-characterized microbial DNA repair MTases.     

Gene transfer to suppress bone marrow alkylation sensitivity

Alkylating agents represent a highly cytotoxic class of chemotherapeutic compounds that are extremely effective anti-tumor agents. Unfortunately, alkylating agents damage both malignant and non-malignant tissues. Bone marrow is especially sensitive to damage by alkylating agent chemotherapy, and is a dose-limiting tissue when treating cancer patients. One strategy to overcome bone marrow sensitivity to alkylating agent exposure involves gene transfer of the DNA repair protein O(6)-methylguanine DNA methyltransferase (O(6)MeG DNA MTase) into bone marrow cells. O(6)MeG DNA MTase is of particular interest because it functions to protect against the mutagenic, clastogenic and cytotoxic effects of many chemotherapeutic alkylating agents. By increasing the O(6)MeG DNA MTase repair capacity of bone marrow cells, it is hoped that this tissue will become alkylation resistant, thereby increasing the therapeutic window for the selective destruction of malignant tissue. In this review, the field of O(6)MeG DNA MTase gene transfer into bone marrow cells will be summarized with an emphasis placed on strategies used for suppressing the deleterious side effects of chemotherapeutic alkylating agent treatment.     

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.     

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.     

Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli.

Escherichia coli ada ogt mutants, which are totally deficient in O6-methylguanine-DNA methyltransferases, have an increased spontaneous mutation rate. This phenotype is particularly evident in starving cells and suggests the generation of an endogenous DNA alkylating agent under this growth condition. We have found that in wild-type cells, the level of the inducible Ada protein is 20-fold higher in stationary-phase and starving cells than in rapidly growing cells, thus enhancing the defense of these cells against DNA damage. The increased level of Ada in stationary cells is dependent on RpoS, a stationary-phase-specific sigma subunit of RNA polymerase. We have also identified a potential source of the mutagenic agent. Nitrosation of amides and related compounds can generate directly acting methylating agents and can be catalyzed by bacteria] enzymes. E. coli moa mutants, which are defective in the synthesis of a molybdopterin cofactor required by several reductases, are deficient in nitrosation activity. It is reported here that a moa mutant shows reduced generation of a mutagenic methylating agent from methylamine (or methylurea) and nitrite added to agar plates. Moreover, a moa mutation eliminates much of the spontaneous mutagenesis in ada ogt mutants. These observations indicate that the major endogenous mutagen is not S-adenosylmethionine but arises by bacterially catalyzed nitrosation.     

Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents.

When Esherichia coli cells are exposed to a low level of simple alkylating agents, they induce the adaptive response which renders them more resistant to the killing and the mutagenic effects of the same or other alkylating agents. This paper describes the isolation of one strain that was deficient in mutagenic adaptation and five that were deficient in both mutagenic and killing adaptation, confirming previous suggestions that killing and mutagenic adaptation are, at least to some extent, separable. These six strains have been called Ada mutants. They were more sensitive to the killing and mutagenic effects of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) than the unadapted Ada+ parent. Thus, the adaptation pathway is responsible for circumventing some alkylation-induced damage even in cells that are preinduced. The increase in mutation frequency seen in Ada cells treated with MNNG was the same whether the cells were lexA+ or lexA, showing that the extra mutations found in Ada- strains do not depend upon the SOS pathway. Ada strains accumulated more O6-methyl guanine lesions than the Ada+ parent on prolonged exposure to MNNG, and this supports the idea that O6-methyl guanine is the most important lesion for MNNG-induced mutagenesis. The ada mutations have been shown to map in the 47 to 53-min region of the E. coli chromosome.     

The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations.     

Induction of SOS response,cellular efflux and oxidative stress response genes by chlorambucil in DNA repair-deficient Escherichia coli cells (ada,ogt and mutS)

Chlorambucil (CLB) is a bifunctional alkylating drug widely used as an anticancer agent and as an immunosuppressant. It is known to be mutagenic, teratogenic and carcinogenic. The cellular actions of CLB have remained poorly investigated. It is very likely that DNA damage and its repair are the key elements determining the destiny of CLB-exposed cells. We investigated the role of two specific DNA repair pathways involved in CLB-induced mutagenicity and gene expression changes by using Escherichia coli strains lacking either (i) two DNA methyltransferase functions (O(6)-methylguanine-DNA methyltransferase I (ada) and II (ogt)), or (ii) mismatch repair (MutS (mutS)). Mutagenicity was determined as the development of ciproxin and rifampicin resistance and the gene expression changes were assessed using expression profiling of all E. coli 4290 open reading frames (ORFs) by cDNA array. Chlorambucil-induced mutants in mutS cells, implying the importance of mismatch repair in preventing CLB-induced mutations. It also induced mutants in the ada, ogt strain, but to a lesser extent than in the wild-type strain. The simultaneous upregulation of several genes of the SOS response, cellular efflux and oxidative stress response, was demonstrated in both of the DNA repair-deficient strains but not in the wild-type cells. These and our previous results show that single-gene knock-out cells use specific gene regulation strategies to avoid mutations and cell death induced by agents such as chlorambucil.     

Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O(6)-methylguanine DNA-methyltransferase in other organisms

Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.     

Expression of the truncated E. coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents

We have constructed a truncated E. coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells. The transfectant cells expressed the truncated E. coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5. These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.     

Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine.

Escherichia coli has two O6-methylguanine DNA methyltransferases that repair alkylation damage in DNA and are encoded by the ada and ogt genes. The ada gene of E. coli also regulates the adaptive response to alkylation damage. The closely related species Salmonella typhimurium possesses methyltransferase activities but does not exhibit an adaptive response conferring detectable resistance to mutagenic methylating agents. We have previously cloned the ada-like gene of S. typhimurium (adaST) and constructed an adaST-deletion derivative of S. typhimurium TA1535. Unexpectedly, the sensitivity of the resulting strain to the mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was similar to that of the parent strain. In this study, we have cloned and sequenced the ogt-like gene of S. typhimurium (ogtST) and characterized ogtST-deletion derivatives of TA1535. The ogtST mutant was more sensitive than the parent strain to the mutagenicity of MNNG and other simple alkylating agents with longer alkyl groups (ethyl, propyl, and butyl). The adaST-ogtST double mutant had a level of hypersensitivity to these agents similar to that of the ogtST single mutant. The ogtST and the adaST-ogtST mutants also displayed a two to three times higher spontaneous mutation frequency than the parent strain and the adaST mutant. These results indicate that the OgtST protein, but not the AdaST protein, plays a major role in protecting S. typhimurium from the mutagenic action of endogenous as well as exogenous alkylating agents.     

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.     

Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli

A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state.     

DNA mismatch repair-induced double-strand breaks

Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis (PFGE), that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB(Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O(6)-methylguanine (O(6)-meG) base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB(Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly.     

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.