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.     

Human cDNA expressing a functional DNA glycosylase excising 3-methyladenine and 7-methylguanine.

A cDNA expression library from a human cell line was introduced into an E. coli strain deficient in the repair of 3-meAde bases in DNA. E. coli strains deficient in the repair of 3-meAde are unusually sensitive to DNA methylating agents. A plasmid pANPG10 (Alkyl-N-Purine-DNA Glycosylase) was rescued from the library based on its ability to reduce the sensitivity of the mutant strain to methylmethane sulfonate. Crude extracts of the E. coli mutant strain hosting the plasmid pANPG10 release both 3-meAde and 7-meGua from DNA. The longest open reading frame in the sequence codes for a polypeptide of 230 amino acids of molecular weight 25.5 kD, with a pI of 9.1. The derived amino acid sequence of the human 3-meAde-DNA glycosylase has 85% sequence identity with the 3-meAde-DNA glycosylase from rat hepatoma cells.     

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.     

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.     

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases

DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix-hairpin-helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases.     

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.     

DNA damage recognition and repair by 3-methyladenine DNA glycosylase I (TAG)

DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escherichia coli 3-methyladenine DNA glycosylase I (TAG) specifically catalyzes the removal of the cytotoxic lesion 3-methyladenine (3mA). The molecular basis for the enzymatic recognition and removal of 3mA from DNA is currently a matter of speculation, in part owing to the lack of a structure of a 3mA-specific glycosylase bound to damaged DNA. Here, high-resolution crystal structures of Salmonella typhi TAG in the unliganded form and in a ternary product complex with abasic DNA and 3mA nucleobase are presented. Despite its structural similarity to the helix-hairpin-helix superfamily of DNA glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. In contrast to other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site. This is the first structural demonstration that conformational relaxation must occur in the DNA upon base hydrolysis. Together with mutational studies of TAG enzymatic activity, these data provide a model for the specific recognition and hydrolysis of 3mA from DNA.     

Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli.

Double-stranded DNA containing 7-methylguanine residues whose imidazole rings have been opened, i.e. 2,6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine residues, may be prepared by treatment of DNA with dimethyl sulfate followed by prolonged incubation at pH 11.4. These substituted formamidopyrimidine residues are actively removed from DNA by a DNA glycosylase present in E. coli cell extracts. The enz;me shows no apparent cofactor requirement and has a molecular weight of about 30 000. The release of ring-opened 7-methyl-guanine residues is due to a previously unrecognized activity, different from the three known E. coli DNA glycosylases that release uracil, 3-methyladenine, and hypoxanthine from DNA. This enzyme may serve to repair a major secondary alkylation product in DNA. In addition, it may remove nonmethylated purines, whose imidazole rings have been opened, from X-irradiated DNA.     

Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II

By in vitro recombination we have constructed hybrid plasmids which can suppress the increased methylmethane sulfonate sensitivity caused by the alkA1 mutation in Escherichia coli. Since the cloned DNA fragment was mapped at 44 to 45 min of the E. coli K12 genetic map, an area where the alkA gene is located, we conclude that the cloned DNA fragment contains the alkA gene itself but not other gene(s) that suppresses the alkA mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkA codes for a polypeptide whose molecular weight is about 30,000. When cells harboring the alkA+ plasmids were grown in the presence of low doses of a simple alkylating agent (adapted condition), the activity of 3-methyladenine DNA glycosylase II was increased. The enzyme activity was copurified with the Mr 30,000 polypeptide. These results indicate that the alkA gene codes for 3-methyladenine DNA glycosylase II. Taking advantage of overproduction of the alkA protein in adapted cells that harbor multicopy plasmids carrying the alkA+ gene, 3-methyladenine DNA glycosylase II has been purified to apparent physical homogeneity.     

Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II

Summary The presence of polydisperse small circular DNAs in wheat cells was first confirmed by the mica-pressadsorption (MPA) method for electron microscopy. To identify their location in the cell, chloroplast and mitochondrial fractions were examined separately by the same method; small circular DNAs were scarcely found in the former but abundantly in the latter fraction, indicating their origin from mitochondria. The size varied greatly, ranging from 0.1 to 2.0 m in contour length. To verify the present finding, the same mitochondrial fraction was examined by the conventional cytochrome-spreading method by which the presence of the same size-class of circular DNAs was confirmed.To know the relationship between the small circular DNAs and cytoplasmic differentiation observed among Tritium (wheat) and Aegilops species, protoplasts isolated from seven alloplasmic lines of common wheat with different cytoplasms were examined by the MPA method. Similar polydisperse small circular DNAs, ranging from 0.1 to 2.5 m in contour length Dere found in all lines, and no clear size differences were noticed among the DNA populations from the cytoplasms of eight Triticum and Aegilops species.     

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.     

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.     

A novel zinc snap motif conveys structural stability to 3-methyladenine DNA glycosylase I

The Escherichia coli 3-methyladenine DNA glycosylase I (TAG) is a DNA repair enzyme that excises 3-methyladenine in DNA and is the smallest member of the helix-hairpin-helix (HhH) superfamily of DNA glycosylases. Despite many studies over the last 25 years, there has been no suggestion that TAG was a metalloprotein. However, here we establish by heteronuclear NMR and other spectroscopic methods that TAG binds 1 eq of Zn2+ extremely tightly. A family of refined NMR structures shows that 4 conserved residues contributed from the amino- and carboxyl-terminal regions of TAG (Cys4, His17, His175, and Cys179) form a Zn2+ binding site. The Zn2+ ion serves to tether the otherwise unstructured amino- and carboxyl-terminal regions of TAG. We propose that this unexpected "zinc snap" motif in the TAG family (CX(12-17)HX(approximately 150)HX(3)C) serves to stabilize the HhH domain thereby mimicking the functional role of protein-protein interactions in larger HhH superfamily members.     

Physical and functional interactions between 3-methyladenine DNA glycosylase and topoisomerase I in mycobacteria

DNA glycosylases play important roles in DNA repair in a variety of organisms, including humans. However, the function and regulation of these enzymes in the pathogenic bacterium Mycobacterium tuberculosis and related species are poorly understood. In the present study, the physical and functional interactions between 3-methyladenine DNA glycosylase (MAG) and topoisomerase I (TopA) in M. tuberculosis and M. smegmatis were characterized. MAG was found to inhibit the function of TopA in relaxing supercoiled DNA. In contrast, TopA stimulated the cleavage function of MAG on a damaged DNA substrate that contains hypoxanthine. The interaction between the two proteins was conserved between the two mycobacterial species. Several mutations in MAG that led to the loss of its interaction with and activity regulation of TopA were also characterized. The results of this study further elucidate glycosylase regulation in both M. smegmatis and M. tuberculosis.     

Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli

We constructed a recombinant plasmid carrying a gene that suppresses tag mutation. To overproduce its gene product, a 0.8-kilobase DNA fragment which carries the gene was placed under the control of the lac promoter in pUC8. 3-Methyladenine-DNA glycosylase activity in cells carrying such plasmids (pCY5) was 450-fold higher than that of wild type strain, on exposure to isopropyl-beta-D-thiogalactopyranoside. From an extract of such cells, the enzyme was purified to apparent physical homogeneity, and the amino acid composition and the amino-terminal amino acid sequence of the enzyme were determined. The data were in accord with nucleotide sequence of the gene, determined by the dideoxy method. It was deduced that 3-methyladenine-DNA glycosylase I comprises 187 amino acids and its molecular weight is 21,100, consistent with the value estimated from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. Only 3-methyladenine was excised from methylated DNA by the purified glycosylase. These results show that the tag is the structural gene for 3-methyladenine-DNA glycosylase I.     

Expression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.

In order to investigate the importance of 3-methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3-methyladenine DNA glycosylase-1 which specifically removes 3-alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus-packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3-methyladenine DNA glycosylase activity in cell extracts showed an up to 10-fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N-methyl-N-nitrosourea than their parent cell lines, indicating that 3-methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3-methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.     

Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase. We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations. The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated. The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini. The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively). On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate. Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT). Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values.     

Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) protects the genome by removing mutagenic uracil residues resulting from deamination of cytosine. Uracil binds in a rigid pocket at the base of the DNA-binding groove of human UDG and the specificity for uracil over the structurally related DNA bases thymine and cytosine is conferred by shape complementarity, as well as by main chain and Asn204 side chain hydrogen bonds. Here we show that replacement of Asn204 by Asp or Tyr147 by Ala, Cys or Ser results in enzymes that have cytosine-DNA glycosylase (CDG) activity or thymine-DNA glycosylase (TDG) activity, respectively. CDG and the TDG all retain some UDG activity. CDG and TDG have kcat values in the same range as typical multisubstrate-DNA glycosylases, that is at least three orders of magnitude lower than that of the highly selective and efficient wild-type UDG. Expression of CDG or TDG in Escherichia coli causes 4- to 100-fold increases in the yield of rifampicin-resistant mutants. Thus, single amino acid substitutions in UDG result in less selective DNA glycosylases that release normal pyrimidines and confer a mutator phenotype upon the cell. Three of the four new pyrimidine-DNA glycosylases resulted from single nucleotide substitutions, events that may also happen in vivo.