Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation.

The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.     

Immunological detection of O6-methylguanine in alkylated DNA

Antibodies to O6-methyldeoxyguanosine were produced in rabbits and utilized in a radioimmunoassay to detect this nucleoside at picomole levels. The specificity of the antibodies was demonstrated by the use of nucleoside analogues as inhibitors in the radioimmunoassay. The antibodies cross-reacted with O6-methylguanosine, O6-methylguanine, and O6-ethylguanosine. There was 10(4) to 10(6) times less sensitivity to inhibition by deoxyadenosine, deoxyguanosine, and guanosine than by O6-methyldeoxyguanosine. The radioimmunoassay also detected O6-methylguanine in DNA alkylated by agents known to produce O6-methylguanine, such as N'-methyl-N-nitrosourea. DNA alkylated with dimethyl sulfate, which does not produce O6-methylguanine in DNA, cross-reacted with the antibodies to a very limited extent. Such an assay system for modified nucleic acid components would be very useful in following the production, persistence, and repair of these lesions in a variety of cells and tissues treated with a broad spectrum of carcinogens and suspected carcinogens.     

Alternative pathways for the in vivo repair of O6-alkylguanine and O4-alkylthymine in Escherichia coli: the adaptive response and nucleotide excision repair.

The in vivo removal of three different O-alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for O6-methylguanine, O6-ethylguanine and O4-ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, O6-methylguanine, O6-ethylguanine and O4-ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild-type E. coli the adaptive response began to contribute to O6-methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of O6-ethylguanine and O4-ethylthymine in wild-type E. coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three O-alkylated lesions was virtually absent in ada- uvr- bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of O-alkylated bases was compromised by an ada- or by a uvr- mutation, the bacteria became more sensitive to alkylation induced killing and mutation.     

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.     

The effect of O6-methylguanine DNA adducts on the adenosine nucleotide switch functions of hMSH2-hMSH6 and hMSH2-hMSH3

The human homologs of prokaryotic mismatch repair have been shown to mediate the toxicity of certain DNA damaging agents; cells deficient in the mismatch repair pathway exhibit resistance to the killing effects of several of these agents. Although previous studies have suggested that the human MutS homologs, hMSH2-hMSH6, bind to DNA containing a variety of DNA adducts, as well as mispaired nucleotides, a number of studies have suggested that DNA binding does not correlate with repair activity. In contrast, the ability to process adenosine nucleotides by MutS homologs appears to be fundamentally linked to repair activity. In this study, oligonucleotides containing a single well defined O(6)-methylguanine adduct were used to examine the extent of lesion-provoked DNA binding, single-step ADP --> ATP exchange, and steady-state ATPase activity by hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers. Interestingly, O(6)-methylguanine lesions when paired with either a C or T were found to stimulate ADP --> ATP exchange, as well as the ATPase activity of purified hMSH2-hMSH6, whereas there was no significant stimulation of hMSH2-hMSH3. These results suggest that O(6)-methylguanine uniquely activates the molecular switch functions of hMSH2-hMSH6.     

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.     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Methylated purines formed in DNA by dimethylnitrosamine in rats previously exposed to hepatotoxic and hepatocarcinogenic regimes: effects on the repair of O6-methylguanine

Studies of mammalian systems for the repair of O6-methylguanine in DNA have revealed large differences in the capacities of tissues and cells to perform this function and in the case of rat liver it has been shown that the O6-methylguanine repair system can be stimulated by exposure to hepatotoxic and hepatocarcinogenic regimes. In this report an assessment is made of possible relationships between toxic liver injury, DNA synthesis, cell proliferation and DNA repair by treating Wistar rats with agents selected to provide differing degrees of liver involvement. The effects of long-term (20 week) treatments with acetylaminofluorene (15 mg/kg/day), quinoxaline 1,4-dioxide (10 mg/kg/day), 4-aminobiphenyl-HCl (15 mg/kg/day) and pronethalol (20 mg/kg/day) were assessed, using the same strain of animals in which the original toxicity and carcinogenicity data were obtained. Repair of O6-methylguanine produced in liver DNA by a low, non-toxic dose (2 mg/kg) of [14C]dimethylnitrosamine was increased 3-4-fold throughout the period of treatment with acetylaminofluorene, to a lesser extent by quinoxaline 1,4-dioxide and 4-aminophenyl-HCl and not at all in the case of pronethalol. No evidence was obtained to indicate a direct relationship between O6-methylguanine repair and either the induced hepatotoxicity or the ensuing increased rates of DNA synthesis which occur following exposure to these agents.     

The Escherichia coli O6-methylguanine-DNA methyltransferase does not repair promutagenic O6-methylguanine residues when present in Z-DNA

The repair of O6-methylguanine present in N-methylnitrosourea (MNU)-treated alternating polynucleotides MNU-poly(dG-dC) X poly(dG-dC) and MNU-poly(dG-me5dC) X poly(dG-me5dC] was investigated using O6-methylguanine-DNA methyltransferase purified from Escherichia coli. Both modified polynucleotides are equally good substrates for the DNA methyltransferase when they are in the B-form. The substrate properties of the MNU-treated polynucleotides do not differ from those of MNU-treated DNA. One of these modified polynucleotides, MNU-poly(dG-me5dC) X (dG-me5dC), can adopt the Z-conformation under physiological conditions. The conformational transition of the poly(dG-me5dC) X poly(dG-me5dC) from the B-form to the Z-form was monitored by the modification of its spectroscopic properties and by the specific binding of antibodies raised against Z-DNA. The O6-methylguanine residues are repaired in MNU-poly(dG-me5dC) X poly(dG-me5dC) in B-form. At variance, the conversion of this template to the Z-form completely inhibits the repair of the O6-methylguanine residues. The cooperative transition from the Z- to the B-form of MNU-poly(dG-me5dC) X poly(dG-me5dC), mediated by intercalating drugs such as ethidium bromide, restores the ability of MNU-poly(dG-me5dC) X poly(dG-me5dC) to be substrate for the transferase. These results imply that the promutagenic DNA lesion O6-methylguanine persists in Z-DNA fragments and suggest that DNA conformation modulates the extent of DNA repair and, as a result, plays an important role in determining the mutagenic potency of chemical carcinogens.     

Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase

The O6-methylguanine-DNA methyltransferase of Escherichia coli acts rapidly and stoichiometrically to convert a mutagenic O6-methylguanine residue in DNA to unsubstituted guanine. Even at low protein concentrations and in the absence of any cofactors, the transfer of a methyl group to one of the protein's own cysteine residues occurs in less than 2 s at 37 degrees C. The entire kinetic process can be followed experimentally at 5 degrees C. Formation of S-methylcysteine in the protein is accompanied by loss of activity and accounts for the exceptional suicide kinetics of this enzyme as well as for the sharp saturation of O6-methylguanine repair observed in vivo. The enzyme can remove greater than 98% of the methyl groups from O6-methylguanine present in alkylated DNA, but leaves N-alkylated purines untouched. Single-stranded DNA containing O6-methylguanine is a poor substrate, with the methyl transfer occurring at approximately 0.1% of the rate for duplex DNA. This latter observation may explain the high frequency of mutations induced by alkylating agents at DNA replication forks.     

ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N-METHYL-N-NITROSOUREA AND METHYL METHANESULPHONATE

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N -[¹⁴C]methyl- N -nitrosourea and [¹⁴C]methyl methanesulphonate. The main product with both compounds was 7-methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7-methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O ⁶-methylguanine, accounting for about 12 per cent of the radioactivity present as 7-methylguanine and cytoplasmic RNA contained about half this amount of O ⁶-methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O ⁶-methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1-methyladenine, 3-methyladenine and 3-methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.     

Intermolecular transacylation of phosphatidylethanolamine by a Butyrivibrio sp.

The alkylation of purine bases in DNA of several rat tissues was determined during weekly injections (10 mg/kg) of N-[3H]methyl-N-nitrosourea, a dose schedule known to selectively induce tumours of the nervous system. Each group of animals was killed 1 week after the final injection, and the DNA hydrolysates were analysed by chromatography on Sephadex G-10. After five weekly applications, O6-methylguanine had accumulated in brain DNA to an extent which greatly exceeded that in kidney, spleen and intestine. In the liver, the final O6-methylguanine concentration was less than 1% of that in brain. Between the first and the fifth injection, the O6-methylguanine/7-methylguanine ratio in cerebral DNA increased from 0.28 to 0.68. In addition, 3-methylguanine was found to accumulate in brain DNA whereas in the other organs no significant quantities of this base were detectable. The results are compatible with the hypothesis that O6-alkylation of guanine in DNA plays a major role in the induction of tumours by N-methyl-N-nitrosourea and related carcinogens. The kinetics of the increase of O6-methylguanine in cerebral DNA suggest that there is no major cell fraction in the brain which is capable of excising chemically methylated bases from DNA. This repair deficiency could be a determining factor in the selective induction of nervous-system tumours by N-methyl-N-nitrosourea and other neuro-oncogenic compounds.     

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O(6)-methylguanine due to high E2F1 regulated mismatch repair

Exposure of stem cells to genotoxins may lead to embryonic lethality or teratogenic effects. This can be prevented by efficient DNA repair or by eliminating genetically damaged cells. Using undifferentiated mouse embryonic stem (ES) cells as a pluripotent model system, we compared ES cells with differentiated cells, with regard to apoptosis induction by alkylating agents forming the highly mutagenic and killing DNA adduct O(6)-methylguanine. Upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ES cells undergo apoptosis at much higher frequency than differentiated cells, although they express a high level of the repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Apoptosis induced by MNNG is due to O(6)-methylguanine DNA adducts, since inhibition of MGMT sensitized ES cells. The high sensitivity of ES cells to O(6)-methylating agents is due to high expression of the mismatch repair proteins MSH2 and MSH6 (MutSalpha), which declines during differentiation. High MutSalpha expression in ES cells was related to a high hyperphosphorylated retinoblastoma (ppRb) level and E2F1 activity that upregulates MSH2, causing, in turn, stabilization of MSH6. Non-repaired O(6)-methylguanine adducts were shown to cause DNA double-stranded breaks, stabilization of p53 and upregulation of Fas/CD95/Apo-1 at significantly higher level in ES cells than in fibroblasts. The high apoptotic response of ES cells to O(6)-methylguanine adducts may contribute to reduction of the mutational load in the progenitor population.     

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.     

Liver DNA alkylation after a single carcinogenic dose of dimethylnitrosamine to newborn and adult CFW Swiss mice

N-nitrosodimethylamine N-demethylase activity, DNA alkylation, capacity for O6-methylguanine repair and cell proliferation were measured in livers of newborn and adult CFW mice after a single carcinogenic dose of DMNA. DNA alkylation was found in newborn and adult mouse livers but it was significantly higher in the newborn. 6- and 7-methyl substitutions of guanine were identified by HPLC analysis in newborn and in adult mouse livers. Metabolic 14C incorporation into adenine and guanine was observed only in liver DNA of newborns. O6-methylguanine levels were higher in newborn than adult mice after a single i.p. dose of [14C]DNMA. Liver DNA repair capacity measured as O6-meG-DNA methyltransferase was higher in adults than in newborns. De novo liver DNA synthesis was more inhibited by DMNA pretreatment in newborn than in adult mice. The relationship between these parameters and the greater neonatal liver tumor susceptibility is discussed.     

Relationship of methyl purines produced by MNNG in adenovirus 5 DNA to viral inactivation in repair-deficient (Mer-) human tumor cell strains

Adenovirus 5 treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) has greater plaque-forming ability in cell strains having the Mer+ phenotype than in strains having the Mer- phenotype. MNNG-treated Mer- strains repair the N3-methyladenine (N3MeA) but not the O6-methylguanine (O6MeG) produced in their DNA, while MNNG-treated Mer+ strains repair both of these adducts. The fate of N7-methylguanine (another DNA adduct produced by MNNG) is similar in Mer+ and Mer- strains. We show in this paper that 2.3 +/- 0.4 O6MeG and 1.4 N3MeA per adenovirus genome correlate with one lethal hit when the survival assay is done using Mer- strains as viral hosts. We suggest that O6MeG is the lesion lethal to the virus.     

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.     

Chronic or acute administration of various dialkylnitrosamines enhances the removal of O6-methylguanine from rat liver DNA in vivo

The effect of pretreatment of rats with various symmetrical dialkylnitrosamines on the repair of O⁶-methylguanine produced in liver DNA by a low dose of [¹⁴C]dimethylnitrosamine (DMN) has been examined. DMN, diethylnitrosamine (DEN), dipropylnitrosamine (DPN) or dibutylnitrosamine (DBN) were administered to rats for 14 consecutive weekdays at a daily dose of 5% of the LD₅₀. Animals were given [¹⁴C]DMN 24 h after the last dose and were killed 6 h later. DNA was extracted from the liver and analysed for methylpurine content after mild acid hydrolysis and Sephadex G-10 chromatography. While the amounts of 3-methyladenine and 7-methylguanine were only slightly different from controls, the amounts of O⁶-methylguanine in the DNA of the dialkylnitrosamine pretreated rats were about 30% of those in control rats, indicating a considerable increase in the capacity to repair this base. Liver ribosomal RNA from control and dialkylnitrosamine pretreated rats contained closely similar amounts of O⁶-methylguanine suggesting that the induced enzyme system does not act on this base in ribosomal RNA in vivo. Pretreatment with these dialkylnitrosamines also enhanced the repair of O⁶-methylguanine in liver DNA when they were given as a single dose (50% of the LD₅₀) either 3 or 7 days before the [¹⁴C]DMN. In addition, single low doses of DMN or DEN (5% of the LD₅₀) given either 1 or 6 days before [¹⁴C]DMN increased O⁶-methylguanine repair and the magnitude of the effect after DEN was similar to that produced by the other pretreatment schedules. The possible mechanism(s) of the induction of O⁶-methylguanine repair and its relation to hepatotoxicity, DNA alkylation, carcinogenesis and the adaptive response in Escherichia coli are discussed.     

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.     

Alkylation damage in DNA and RNA--repair mechanisms and medical significance

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.