The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.     

Release of normal bases from intact DNA by a native DNA repair enzyme.

Base excision repair is initiated by DNA glycosylases removing inappropriate bases from DNA. One group of these enzymes, comprising 3-methyladenine DNA glycosylase II (AlkA) from Escherichia coli and related enzymes from other organisms, has been found to have an unusual broad specificity towards quite different base structures. We tested whether such enzymes might also be capable of removing normal base residues from DNA. The native enzymes from E.coli, Saccharomyces cerevisiae and human cells promoted release of intact guanines with significant frequencies, and further analysis of AlkA showed that all the normal bases can be removed. Transformation of E. coli with plasmids expressing different levels of AlkA produced an increased spontaneous mutation frequency correlated with the expression levels, indicating that excision of normal bases occurs at biologically significant rates. We propose that the broad specificity 3-methyladenine DNA glycosylases represent a general type of repair enzyme 'pulling' bases in DNA largely at random, without much preference for a specific structure. The specificity for release of damaged bases occurs because base structure alterations cause instability of the base-sugar bonds. Damaged bases are therefore released more readily than normal bases once the bond activation energy is reduced further by the enzyme. Qualitatively, the model correlates quite well with the relative rate of excision observed for most, if not all, of the substrates described for AlkA and analogues.     

Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase

Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.     

Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli.

Escherichia coli has two DNA glycosylases for repair of DNA damage caused by simple alkylating agents. The inducible AlkA DNA glycosylase (3-methyladenine [m3A] DNA glycosylase II) removes several different alkylated bases including m3A and 3-methylguanine (m3G) from DNA, whereas the constitutively expressed Tag enzyme (m3A DNA glycosylase I) has appeared to be specific for excision of m3A. In this communication we have reexamined the substrate specificity of Tag by using synthetic DNA rich in GC base pairs to facilitate detection of any possible methyl-G removal. In such DNA alkylated with [3H]dimethyl sulphate, we found that m3G was excised from double-stranded DNA by both glycosylases, although more efficiently by AlkA than by Tag. This was further confirmed using both N-[3H]methyl-N-nitrosourea- and [3H]dimethyl sulphate-treated native DNA, from which Tag excised m3G with an efficiency that was about 70 times lower than for AlkA. These results can explain the previous observation that high levels of Tag expression will suppress the alkylation sensitivity of alkA mutant cells, further implying that m3G is formed in quantity sufficient to represent an important cytotoxic lesion if left unrepaired in cells exposed to alkylating agents.     

Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends

The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.     

Solution structure and base perturbation studies reveal a novel mode of alkylated base recognition by 3-methyladenine DNA glycosylase I

The specific recognition mechanisms of DNA repair glycosylases that remove cationic alkylpurine bases in DNA are not well understood partly due to the absence of structures of these enzymes with their cognate bases. Here we report the solution structure of 3-methyladenine DNA glycosylase I (TAG) in complex with its 3-methyladenine (3-MeA) cognate base, and we have used chemical perturbation of the base in combination with mutagenesis of the enzyme to evaluate the role of hydrogen bonding and pi-cation interactions in alkylated base recognition by this DNA repair enzyme. We find that TAG uses hydrogen bonding with heteroatoms on the base, van der Waals interactions with the 3-Me group, and conventional pi-pi stacking with a conserved Trp side chain to selectively bind neutral 3-MeA over the cationic form of the base. Discrimination against binding of the normal base adenine is derived from direct sensing of the 3-methyl group, leading to an induced-fit conformational change that engulfs the base in a box defined by five aromatic side chains. These findings indicate that base specific recognition by TAG does not involve strong pi-cation interactions, and suggest a novel mechanism for alkylated base recognition and removal.     

Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae; a homologue to the bacterial alkA gene.

An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.     

3-Methyladenine DNA glycosylase I is an unexpected helix-hairpin-helix superfamily member

The Escherichia coli enzyme 3-methyladenine DNA glycosylase I (TAG) hydrolyzes the glycosidic bond of 3-methyladenine (3-MeA) in DNA and is found in many bacteria and some higher eukaryotes. TAG shows little primary sequence identity with members of the well-known helix-hairpin-helix (HhH) superfamily of DNA repair glycosylases, which consists of AlkA, EndoIII, MutY and hOGG1. Unexpectedly, the three-dimensional solution structure reported here reveals TAG as member of this superfamily. The restricted specificity of TAG for 3-MeA bases probably arises from its unique aromatic rich 3-MeA binding pocket and the absence of a catalytic aspartate that is present in all other HhH family members.     

DNA damage in human colonic mucosa cells induced by bleomycin and the protective action of vitamin E

Using the alkaline comet assay, we showed that bleomycin at 0.1-5 microg/ml induced DNA strand breaks and/or alkali-labile sites, measurable as the comet tail moment, in human colonic mucosa cells. This DNA damage was completely repaired during a 120-minute post-treatment incubation of the cells. Post-treatment of the bleomycin-damaged DNA with 3-methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage, indicating that the drug could induce alkylative bases in DNA. We did not observe any change in the comet tail moment in the presence of catalase. Vitamin E ((+)-alpha -tocopherol) decreased DNA damage induced by bleomycin. The results obtained suggest that hydrogen peroxide might not be involved in the formation of DNA lesions induced by bleomycin in the colonic mucosa cells.     

Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine, or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet light; low levels of endonuclease activity, obtained when X-irradiated, alkylated, or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.     

Methylated purines in the deoxyribonucleic acid of various Syrian-golden-hamster tissues after administration of a hepatocarcinogenic dose of dimethylnitrosamine.

1. DNA was extracted from livers, kidneys and lungs of Syrian golden hamsters at various times (up to 96h) after injection of a hepatocarcinogenic dose of [14C]dimethylnitrosamine. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. At 7h after dimethylnitrosamine administration liver DNA was alkylated to the greatest extent, followed by that of lung and kidney, the values for which were 8 and 3% respectively of those for liver. 3. The O6-methylguanine/7-methylguanine ratios were initially the same in all three organs and in the liver DNA of rats under similar conditions of dose. 4. O6-Methylguanine was the most persistent alkylated purine in all three hamster tissues. There was evidence for excision of 7-methyl-guanine, the highest activity for this being present in the liver. 5. Detectable amounts of the minor products 3-methyladenine, 1-methyladenine, 3-methylguanine and 7-methyladenine were present in most hamster tissues, and their individual rates of loss from liver DNA were determined. 6. Ring-labelling of the normal purines in DNA was highest in the liver, followed closely by the lung (80% of that in liver) whereas the kidney had very low incorporation (3% of that in liver). 7. The results are discussed with respect to the hepatotoxicity of dimethylnitrosamine, the miscoding potential of the various alkylation products and the induction of liver tumours in hamsters.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.     

Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats

3-methyladenine DNA glycosylases initiate repair of cytotoxic and promutagenic alkylated bases in DNA. We demonstrate by comparative modelling that Bacillus cereus AlkD belongs to a new, fifth, structural superfamily of DNA glycosylases with an alpha-alpha superhelix fold comprising six HEAT-like repeats. The structure reveals a wide, positively charged groove, including a putative base recognition pocket. This groove appears to be suitable for the accommodation of double-stranded DNA with a flipped-out alkylated base. Site-specific mutagenesis within the recognition pocket identified several residues essential for enzyme activity. The results suggest that the aromatic side chain of a tryptophan residue recognizes electron-deficient alkylated bases through stacking interactions, while an interacting aspartate-arginine pair is essential for removal of the damaged base. A structural model of AlkD bound to DNA with a flipped-out purine moiety gives insight into the catalytic machinery for this new class of DNA glycosylases.     

Excision of 8-methylguanine site-specifically incorporated into oligonucleotide substrates by the AlkA protein of Escherichia coli

8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.     

Purification and characterization of 3-methyladenine DNA glycosylase I from Escherichia coli.

We have purified 3-methyladenine DNA glycosylase I from Escherichia coli to apparent physical homogeneity. The enzyme preparation produced a single band of Mr 22,500 upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis in good agreement with the molecular weight deduced from the nucleotide sequence of the tag gene (Steinum, A.-L. and Seeberg, E. (1986) Nucl. Acids Res. 14, 3763-3772). HPLC confirmed that the only detectable alkylation product released from (3H)dimethyl sulphate treated DNA was 3-methyladenine. The DNA glycosylase activity showed a broad pH optimum between 6 and 8.5, and no activity below pH 5 and above pH 10. MgSO4, CaCl2 and MnCl2 stimulated enzyme activity, whereas ZnSO4 and FeCl3 inhibited the enzyme at 2 mM concentration. The enzyme was stimulated by caffeine, adenine and 3-methylguanine, and inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 3-methyladenine. The enzyme showed no detectable endonuclease activity on native, depurinated or alkylated plasmid DNA. However, apurinic sites were introduced in alkylated DNA as judged from the strand breaks formed by mixtures of the tag enzyme and the bacteriophage T4 denV enzyme which has apurinic/apyrimidinic endonuclease activity. It was calculated that wild-type E. coli contains approximately 200 molecules per cell of 3-methyladenine DNA glycosylase I.     

Free radical scavengers can modulate the DNA-damaging action of alloxan

Alloxan can generate diabetes in experimental animals and its action can be associated with the production of free radicals. It is therefore important to check how different substances often referred to as free radical scavengers may interact with alloxan, especially that some of these substance may show both pro- and antioxidant activities. Using the alkaline comet assay we showed that alloxan at concentrations 0.01-50 microM induced DNA damage in normal human lymphocytes in a dose-dependent manner. Treated cells were able to recover within a 120-min incubation. Vitamins C and E at 10 and 50 microM diminished the extent of DNA damage induced by 50 microM alloxan. Pre-treatment of the lymphocytes with a nitrone spin trap, alpha-(4-pyridil-1-oxide)- N-t-butylnitrone (POBN) or ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which mimics glutathione peroxides, reduced the alloxan-evoked DNA damage. The cells exposed to alloxan and treated with formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results confirmed that free radicals are involved in the formation of DNA lesions induced by alloxan. The results also suggest that alloxan can generate oxidized DNA bases with a preference for purines and contribute to their alkylation.     

Enzymatic repair of 5-formyluracil. I. Excision of 5-formyluracil site-specifically incorporated into oligonucleotide substrates by alka protein (Escherichia coli 3-methyladenine DNA glycosylase II).

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. (1)H and (13)C NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.     

Nucleotide sequence of the tag gene from Escherichia coli.

We have determined the complete nucleotide sequence of the tag gene, encoding 3-methyladenine DNA glycosylase I from Escherichia coli. From the nucleotide sequence it is deduced that the tag enzyme consists of 187 amino-acids and has a calculated molecular weight of 21.1 kdaltons. The tag enzyme is unusually rich in cysteine (8 residues) with a cluster of three consecutive cysteines near the C-terminal end. The tag coded DNA glycosylase does not show significant sequence homology to the alkA coded glycosylase in spite of that both of these enzymes catalyze the release of free 3-methyladenine from alkylated DNA.