Secondary literature sources for ENDO3c

The following references were automatically generated.

Eide L, Fosberg E, Hoff E, Seeberg E
Overexpression of endonuclease III protects Escherichia coli mutants defective in alkylation repair against lethal effects of methylmethanesulphonate.
FEBS Lett. 2001; 491: 59-62
Display abstract
Endonuclease III of Escherichia coli is normally involved in the repair of oxidative DNA damage. Here, we have investigated a possible role of EndoIII in the repair of alkylation damage because of its structural similarity to the alkylation repair enzyme 3-methyladenine DNA glycosylase II. It was found that overproduction of EndoIII partially relieved the alkylation sensitivity of alkA mutant cells. Site-directed mutagenesis to make the active site of EndoIII more similar to AlkA (K120W) had an adverse effect on the complementation and the mutant protein apparently inhibited repair by competing for the substrate without base release. These results suggest that EndoIII might replace AlkA in some aspect of alkylation repair, although high expression levels are needed to produce this effect.

Marenstein DR et al.
Stimulation of human endonuclease III by Y box-binding protein 1 (DNA-binding protein B). Interaction between a base excision repair enzyme and a transcription factor.
J Biol Chem. 2001; 276: 21242-9
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Human endonuclease III (hNth1) is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation. Using duplex 2'-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are also members of the endonuclease III family. This difference in rates contrasts with the equality of rates found in Escherichia coli and Saccharomyces cerevisiae endonuclease III homologs. A yeast two-hybrid screen for potential modulators of hNth1 activity revealed interaction with the damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified as DNA-binding protein B (DbpB). The in vitro addition of His(6)YB-1 to hNth1 increased the rate of DNA glycosylase and AP lyase activity. Analysis revealed that YB-1 affects the steady state equilibrium between the covalent hNth1-AP site Schiff base ES intermediate and the noncovalent ES intermediate containing the AP aldehydic sugar and the epsilon-amino group of the hNth1 active site lysine. This equilibrium may be a checkpoint in modulating hNth1 activity.

Sandler SJ, Marians KJ
Role of PriA in replication fork reactivation in Escherichia coli.
J Bacteriol. 2000; 182: 9-13

Plochocka D, Kierzek A, Obtulowicz T, Tudek B, Zielenkiewicz P
3-Methyladenine-DNA glycosylase I from Escherichia coli-computer modeling and supporting experimental evidence.
Biochem Biophys Res Commun. 2000; 268: 724-7
Display abstract
TagA (3-methyladenine-DNA glycosylase I) excises 3-methyadenine and 3-methylguanine from alkylated DNA. The structure of this enzyme has not yet been determined experimentally. We propose a three-dimensional model of the TagA protein based on the threading algorithm. The model shows that TagA is a mostly alpha-helical protein, in agreement with circular dichroism measurements. None of the eight cysteines present in the TagA sequence forms a disulfide bridge in the model structure, which has also been experimentally verified with the use of Ellman method.

Sugahara M et al.
Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8.
EMBO J. 2000; 19: 3857-69
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The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N-glycosylase activity) and cleaves both the 3'- and 5'-phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 A resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn(2+) ion of the zinc finger. MutM is composed of two distinct and novel domains connected by a flexible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three-dimensional structure and taking account of previous biochemical experiments, we propose a DNA-binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA-binding motifs (the zinc finger and the helix-two-turns-helix motifs) suggest that the oxidized base is flipped out from double-stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Verhoeven EE, van Kesteren M, Moolenaar GF, Visse R, Goosen N
Catalytic sites for 3' and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC.
J Biol Chem. 2000; 275: 5120-3
Display abstract
Nucleotide excision repair in Escherichia coli is a multistep process in which DNA damage is removed by incision of the DNA on both sides of the damage, followed by removal of the oligonucleotide containing the lesion. The two incision reactions take place in a complex of damaged DNA with UvrB and UvrC. It has been shown (Lin, J. -J., and Sancar, A. (1992) J. Biol. Chem. 267, 17688-17692) that the catalytic site for incision on the 5' side of the damage is located in the UvrC protein. Here we show that the catalytic site for incision on the 3' side is in this protein as well, because substitution R42A abolishes 3' incision, whereas formation of the UvrBC-DNA complex and the 5' incision reaction are unaffected. Arg(42) is part of a region that is homologous to the catalytic domain of the homing endonuclease I-TevI. We propose that the UvrC protein consists of two functional parts, with the N-terminal half for the 3' incision reaction and the C-terminal half containing all the determinants for the 5' incision reaction.

Theis K, Skorvaga M, Machius M, Nakagawa N, Van Houten B, Kisker C
The nucleotide excision repair protein UvrB, a helicase-like enzyme with a catch.
Mutat Res. 2000; 460: 277-300
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Nucleotide excision repair (NER) is a universal DNA repair mechanism found in all three kingdoms of life. Its ability to repair a broad range of DNA lesions sets NER apart from other repair mechanisms. NER systems recognize the damaged DNA strand and cleave it 3', then 5' to the lesion. After the oligonucleotide containing the lesion is removed, repair synthesis fills the resulting gap. UvrB is the central component of bacterial NER. It is directly involved in distinguishing damaged from undamaged DNA and guides the DNA from recognition to repair synthesis. Recently solved structures of UvrB from different organisms represent the first high-resolution view into bacterial NER. The structures provide detailed insight into the domain architecture of UvrB and, through comparison, suggest possible domain movements. The structure of UvrB consists of five domains. Domains 1a and 3 bind ATP at the inter-domain interface and share high structural similarity to helicases of superfamilies I and II. Not related to helicase structures, domains 2 and 4 are involved in interactions with either UvrA or UvrC, whereas domain 1b was implicated for DNA binding. The structures indicate that ATP binding and hydrolysis is associated with domain motions. UvrB's ATPase activity, however, is not coupled to the separation of long DNA duplexes as in helicases, but rather leads to the formation of the preincision complex with the damaged DNA substrate. The location of conserved residues and structural comparisons with helicase-DNA structures suggest how UvrB might bind to DNA. A model of the UvrB-DNA interaction in which a beta-hairpin of UvrB inserts between the DNA double strand has been proposed recently. This padlock model is developed further to suggest two distinct consequences of domain motion: in the UvrA(2)B-DNA complex, domain motions lead to translocation along the DNA, whereas in the tight UvrB-DNA pre-incision complex, they lead to distortion of the 3' incision site.

Lee CS
Excision repair of 2,5-diaziridinyl-1,4-benzoquinone (DZQ)-DNA adduct by bacterial and mammalian 3-methyladenine-DNA glycosylases.
Mol Cells. 2000; 10: 723-7
Display abstract
The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.

Hollis T, Lau A, Ellenberger T
Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase.
Mutat Res. 2000; 460: 201-10
Display abstract
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.

Hollis T, Ichikawa Y, Ellenberger T
DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA.
EMBO J. 2000; 19: 758-66
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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.

Ingleston SM, Sharples GJ, Lloyd RG
The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome.
EMBO J. 2000; 19: 6266-74
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Holliday junctions are four-way branched DNA structures formed during recombination, replication and repair. They are processed in Escherichia coli by the RuvA, RuvB and RuvC proteins. RuvA targets the junction and facilitates loading of RuvB helicase and RuvC endonuclease to form complexes that catalyse junction branch migration (RuvAB) and resolution (RuvABC). We investigated the role of RuvA in these reactions and in particular the part played by the acidic pin located on its DNA-binding surface. By making appropriate substitutions of two key amino acids (Glu55 and Asp56), we altered the charge on the pin and investigated how this affected junction binding and processing. We show that two negative charges on each subunit of the pin are crucial. They facilitate junction targeting by preventing binding to duplex DNA and also constrain branch migration by RuvAB in a manner critical for junction processing. These findings provide the first direct evidence that RuvA has a mechanistic role in branch migration. They also provide insight into the coupling of branch migration and resolution by the RuvABC resolvasome.

Zharkov DO et al.
Role for lysine 142 in the excision of adenine from A:G mispairs by MutY DNA glycosylase of Escherichia coli.
Biochemistry. 2000; 39: 14768-78
Display abstract
MutY participates in the repair of oxidatively damaged DNA by excising adenine from dA:dG and dA:8-oxodG mispairs; this DNA glycosylase can be cross-linked to DNA through Lys-142. We have investigated the properties of a mutant protein in which Lys-142 is replaced by glutamine. Using the rifampicin resistance assay, MutY K142Q was shown to complement the mutY mutator phenotype to the same extent as wild-type MutY. Although MutY K142Q does not form a Schiff base with DNA, it retains in part the catalytic properties of wild-type enzyme. The K142Q mutation selectively impairs processing of DNA containing dA:dG mispairs but not that of substrates containing dA:8-oxodG. Decreased substrate processing is mediated primarily via an increase in K(D) (21.8 nM for MutY vs 298 nM for MutY K142Q). The catalytic constant, measured in single turnover experiments, was not significantly affected. At pH < 6.0, the activity of MutY K142Q on the dA:dG mispair was approximately the same as for wild-type protein, suggesting that a dG(anti) to dG(syn) transition is effected at low pH. The three-dimensional structure of the catalytic domain of MutY K142Q, determined at 1.35 A resolution, shows no significant differences between wild-type and mutant protein, indicating that Lys-142 is not critical for maintaining the conformation of MutY. We conclude that Lys-142 recognizes guanine in the dA:dG mispair, helping position this residue in the syn conformation and facilitating binding of substrate DNA. Lys-142 is not involved in the catalytic steps of base excision.

Masaoka A, Terato H, Kobayashi M, Honsho A, Ohyama Y, Ide H
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).
J Biol Chem. 1999; 274: 25136-43
Display abstract
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.

Putnam CD et al.
Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase.
J Mol Biol. 1999; 287: 331-46
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Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi). A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity. High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi. The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes. Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA. Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex. Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands. A similar beta-zipper is observed in the conversion between the open and closed forms of UDG. The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity.

Lutsenko E, Bhagwat AS
The role of the Escherichia coli mug protein in the removal of uracil and 3,N(4)-ethenocytosine from DNA.
J Biol Chem. 1999; 274: 31034-8
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The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U.G mismatches (Gallinari, P., and Jiricny, J. (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug). It has also been described to remove 3,N(4)-ethenocytosine (epsilonC) from epsilonC.G mismatches (Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8508-8513). We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E. coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U.G and T.G mismatches, respectively, we conclude that Mug does not repair U.G or T.G mismatches in vivo. A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E. coli. Cell-free extracts from mug(+) ung cells show very little ability to remove uracil from DNA, but can excise epsilonC. The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E. coli that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde.

Brooks N, McHugh PJ, Lee M, Hartley JA
The role of base excision repair in the repair of DNA adducts formed by a series of nitrogen mustard-containing analogues of distamycin of increasing binding site size.
Anticancer Drug Des. 1999; 14: 11-8
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The role of base excision repair in the repair of alkylation damage produced by a series of sequence specific oligopyrrole-containing analogues of distamycin A that tether benzoic acid mustard (BAM) has been examined. Whereas BAM alkylates and cross-links in the major groove of DNA, attachment to pyrrole units produces monoalkylations in the minor groove of DNA at AT tracts. Both sequence specificity of alkylation and cytotoxicity increase from one to three attached pyrrole units (compounds 1-3), and with 3 alkylation is selective for purine-N3 in the sequence 5'-TTTTGPu (where Pu = guanine or adenine). In a model bacterial (Escherichia coli) system repair of the sequence specific minor groove alkylations produced by 2 and 3 does not appear to involve BER, since neither a formamidopyrimidine-DNA glycosylase repair deficient E. coli mutant (BH 20, fpg- mutant) nor a 3-methyladenine-DNA glycosylase repair deficient mutant (GC 4803, tag-alkA- mutant) showed increased cytotoxicity to 2 or 3 compared with the wild type, AB 1157. The monopyrrole compound 1 was, however, approximately 4-fold more cytotoxic to the GC 4803 mutant compared with wild type and BH 20, suggesting a role for the 3-methyladenine-DNA glycosylase in the recognition and excision of the adducts formed by 1. In contrast, increased sensitivity (> 10-fold) was observed for the conventional nitrogen mustard BAM in the BH 20 strain, suggesting a role for the formamidopyrimidine-DNA glycosylase in the repair of the lesions produced by the agent. In a cell-free system the E. coli 3-methyladenine-DNA glycosylase (AlkA) was shown to remove alkylations at 5'-TTTTGPu sequences. However, the efficiency in removing the adducts formed by the oligopyrrole compounds decreased dramatically from compound 1 to compound 3. Increasing the size of the DNA adduct formed in the minor groove therefore decreased the efficiency of recognition and removal of the adduct by the DNA glycosylase.

Williams SD, David SS
Formation of a Schiff base intermediate is not required for the adenine glycosylase activity of Escherichia coli MutY.
Biochemistry. 1999; 38: 15417-24
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The mutY gene product of Escherichia coli is a 39-kDa protein that catalyzes the removal of adenine bases mispaired with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. Although adenine removal proceeds via monofunctional glycosylase activity, MutY is able to form covalent adducts with substrate DNA in the presence of borohydride, a trait otherwise known to be associated only with enzymes having bifunctional glycosylase/AP lyase activity. To help identify active site residues involved in the formation of MutY-DNA adducts in the presence of borohydride, a series of site-directed mutant forms of MutY were generated. Our data show that Lys 142 is the primary residue involved in cross-link formation. The absence of Lys 142 results in near elimination of the enzyme-DNA adducts formed relative to wild-type, suggesting that this residue is the primary one involved in forming covalent associations with DNA during MutY catalysis. Importantly, the enzymatic activity and DNA binding of the K142A enzyme is nearly identical to the WT enzyme. This shows that formation of the covalent intermediate is not required for adenine removal by MutY. Furthermore, this suggests that the covalent intermediate is formed by reaction of Lys 142 with the OG/G:(AP site) product, and this may be a consequence of MutY's unusually high affinity for the product of its glycosylase action.

Begley TJ, Haas BJ, Noel J, Shekhtman A, Williams WA, Cunningham RP
A new member of the endonuclease III family of DNA repair enzymes that removes methylated purines from DNA.
Curr Biol. 1999; 9: 653-6
Display abstract
DNA is constantly exposed to endogenous andexogenous alkylating agents that can modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2]. Alkylation damage is removed by the action of DNA glycosylases, which initiate the base excision repair pathway and protect the sequence information of the genome [3-5]. We have identified a new class of methylpurine DNA glycosylase, designated MpgII, that is a member of the endonuclease III family of DNA repair enzymes. We expressed and purified MpgII from Thermotoga maritima and found that the enzyme releases both 7-methylguanine and 3-methyladenine from DNA. We cloned the MpgII genes from T. maritima and from Aquifex aeolicus and found that both genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli alkA tagA double mutants, which are deficient in the repair of alkylated bases. Analogous genes are found in other Bacteria and Archaea and appear to be the only genes coding for methylpurine DNA glycosylase activity in these organisms. MpgII is the fifth member of the endonuclease III family of DNA repair enzymes, suggesting that the endonuclease III protein scaffold has been modified during evolution to recognize and repair a variety of DNA damage.

Xiao G, Tordova M, Jagadeesh J, Drohat AC, Stivers JT, Gilliland GL
Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.
Proteins. 1999; 35: 13-24
Display abstract
The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.

Barrett TE et al.
Crystal structure of a thwarted mismatch glycosylase DNA repair complex.
EMBO J. 1999; 18: 6599-609
Display abstract
The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine. Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.

Posnick LM, Samson LD
Imbalanced base excision repair increases spontaneous mutation and alkylation sensitivity in Escherichia coli.
J Bacteriol. 1999; 181: 6763-71
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Inappropriate expression of 3-methyladenine (3MeA) DNA glycosylases has been shown to have harmful effects on microbial and mammalian cells. To understand the underlying reasons for this phenomenon, we have determined how DNA glycosylase activity and substrate specificity modulate glycosylase effects in Escherichia coli. We compared the effects of two 3MeA DNA glycosylases with very different substrate ranges, namely, the Saccharomyces cerevisiae Mag1 and the E. coli Tag glycosylases. Both glycosylases increased spontaneous mutation, decreased cell viability, and sensitized E. coli to killing by the alkylating agent methyl methanesulfonate. However, Tag had much less harmful effects than Mag1. The difference between the two enzymes' effects may be accounted for by the fact that Tag almost exclusively excises 3MeA lesions, whereas Mag1 excises a broad range of alkylated and other purines. We infer that the DNA lesions responsible for changes in spontaneous mutation, viability, and alkylation sensitivity are abasic sites and secondary lesions resulting from processing abasic sites via the base excision repair pathway.

Shirakawa M
[Structure and function of DNA repair factors]
Tanpakushitsu Kakusan Koso. 1999; 44: 485-94

Messer W et al.
Functional domains of DnaA proteins.
Biochimie. 1999; 81: 819-25
Display abstract
Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

Barrett TE et al.
Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions.
Cell. 1998; 92: 117-29
Display abstract
G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA. Uracil is removed in a base-excision repair pathway by uracil DNA-glycosylase (UDG), which excises uracil from both single- and double-stranded DNA. Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both uracil and thymine, but only from mispairs with guanine. Crystal structures of the mismatch-specific uracil DNA-glycosylase (MUG) from E. coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity. Details of the MUG structure explain its thymine DNA-glycosylase activity and the specificity for G:U/T mispairs, which derives from direct recognition of guanine on the complementary strand.

Rafferty JB et al.
Structural similarities between Escherichia coli RuvA protein and other DNA-binding proteins and a mutational analysis of its binding to the holliday junction.
J Mol Biol. 1998; 278: 105-16
Display abstract
Comparison of the structure of Escherichia coli RuvA with other proteins in the Protein Data Bank gives insights into the probable modes of association of RuvA with the Holliday junction during homologous recombination. All three domains of the RuvA protein possess striking structural similarities to other DNA-binding proteins. Additionally, the second domain of RuvA contains two copies of the helix-hairpin-helix (HhH) structural motif, which has been implicated in non-sequence-specific DNA binding. The two copies of the motif are related by approximate 2-fold symmetry and may form a bidentate DNA-binding module. The results described provide support for the organization of the arms of the DNA in our RuvA/Holliday junction complex model and support the involvement of the HhH motifs in DNA binding.

Lau AY, Scharer OD, Samson L, Verdine GL, Ellenberger T
Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision.
Cell. 1998; 95: 249-58
Display abstract
DNA N-glycosylases are base excision-repair proteins that locate and cleave damaged bases from DNA as the first step in restoring the genetic blueprint. The human enzyme 3-methyladenine DNA glycosylase removes a diverse group of damaged bases from DNA, including cytotoxic and mutagenic alkylation adducts of purines. We report the crystal structure of human 3-methyladenine DNA glycosylase complexed to a mechanism-based pyrrolidine inhibitor. The enzyme has intercalated into the minor groove of DNA, causing the abasic pyrrolidine nucleotide to flip into the enzyme active site, where a bound water is poised for nucleophilic attack. The structure shows an elegant means of exposing a nucleotide for base excision as well as a network of residues that could catalyze the in-line displacement of a damaged base from the phosphodeoxyribose backbone.

Tudek B, Van Zeeland AA, Kusmierek JT, Laval J
Activity of Escherichia coli DNA-glycosylases on DNA damaged by methylating and ethylating agents and influence of 3-substituted adenine derivatives.
Mutat Res. 1998; 407: 169-76
Display abstract
Methylating and ethylating agents are used in the chemical industry and produced during tobacco smoking. They generate DNA base damage whose role in cancer induction has been documented. Alkylated bases are repaired by the base excision repair pathway. We have established the repair efficiency of methylated and ethylated bases by various Escherichia coli repair proteins, namely 3-methyladenine-DNA-glycosylase I (TagA protein), which excises 3-methyladenine and 3-methylguanine, 3-methyladenine-DNA-glycosylase II (AlkA protein), which has a broad substrate specificity including 3- and 7-alkylated purines and the formamidopyrimidine(Fapy)-DNA-glycosylase (Fpg protein) repairing imidazole ring-opened 7-methylguanine. The comparison of the Km values of these various enzymes showed that methylated bases were excised more efficiently than ethylated bases. Several 3-alkyladenine derivatives have been synthesized and examined for their ability to inhibit the activity of the various repair proteins. We have shown that 3-ethyl-, 3-propyl-, 3-butyl- and 3-benzyladenine were much more efficient inhibitors of TagA protein than 3-methyladenine. The inhibitory effect was increased with the increase of the size of alkyl-group and IC50 for 3-benzyladenine was 0.4 +/- 0.1 microM as compared to 1.5 +/- 0.3 mM for 3-methyladenine. These compounds inhibited neither the AlkA protein nor human 3-methyladenine-DNA-glycosylase (ANPG protein). Moreover, 3-hydroxyethyladenine did not affect the activity of any of these enzymes. Taken together, these results suggest that hydrophobic interactions are involved in the mechanism of inhibition and/or recognition and excision of alkylated purines by TagA protein.

Borys E, Kusmierek JT
Endogenous and exogenous DNA lesions recognized by N-alkylpurine-DNA glycosylases.
Acta Biochim Pol. 1998; 45: 579-86
Display abstract
The combined action of glycosylases and abasic site-specific endonucleases on damaged bases in DNA results in single strand breaks.In plasmid DNA, as a consequence, the covalently closed circular (ccc) form is converted to the open circular (oc) form, and this can be quantitated by agarose gel electrophoresis. We studied DNA lesions sensitive to E. coli 3-methyladenine-DNA glycosylase II (AlkA) and cloned human N-alkylpurine-DNA glycosylase (ANPG-40) which are known to excise alkylated bases and etheno adducts. pBR322 and pAlk10 plasmids not pretreated with mutagens were cleaved by both glycosylases in the presence of enzymes possessing endonucleolytic activity, which indicates that plasmids contain unknown, endogenously formed adducts. Plasmids pretreated with chloroacetaldehyde, a mutagen forming etheno adducts, exhibited enhanced sensitivity to both glycosylases. Adducts formed by acrolein and croton aldehyde were excised by AlkA, but not by ANPG-40, whereas malondialdehyde adducts were not excised by either glycosylase. Bulky p-benzochinone adducts were not excised by AlkA, however, the plasmid pretreated with this mutagen was incised by endonucleases, possibly without prior generation of an abasic site. These examples show that examination of conformational changes of plasmid DNA can be taken advantage of to study the specificity of N-alkylpurine-DNA-glycosylases.

Zharkov DO, Grollman AP
MutY DNA glycosylase: base release and intermediate complex formation.
Biochemistry. 1998; 37: 12384-94
Display abstract
MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety. We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme. MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage. The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates. The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases. MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY. The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break. Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.

Berdal KG, Johansen RF, Seeberg E
Release of normal bases from intact DNA by a native DNA repair enzyme.
EMBO J. 1998; 17: 363-7
Display abstract
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.

Mizuno T, Tanaka I
Structure of the DNA-binding domain of the OmpR family of response regulators.
Mol Microbiol. 1997; 24: 665-7

Yamamoto K, Uraki F, Yonei S, Yukawa O
Enzymatic repair mechanisms for base modifications induced by oxygen radicals.
J Radiat Res (Tokyo). 1997; 38: 1-4

Zharkov DO, Rieger RA, Iden CR, Grollman AP
NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein.
J Biol Chem. 1997; 272: 5335-41
Display abstract
Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.

Zeng Q, Summers AO
A glutamate uptake regulatory protein (Grp) in Escherichia coli?
Mol Microbiol. 1997; 24: 231-2

Gogos A, Cillo J, Clarke ND, Lu AL
Specific recognition of A/G and A/7,8-dihydro-8-oxoguanine (8-oxoG) mismatches by Escherichia coli MutY: removal of the C-terminal domain preferentially affects A/8-oxoG recognition.
Biochemistry. 1996; 35: 16665-71
Display abstract
Escherichia coli MutY is a 39 kDa adenine DNA glycosylase and 3' apurinic/apyrimidinic (AP) lyase that is active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. 8-oxoG (7,8-dihydro-8-oxoguanine or GO) is a major stable product of oxidative damage, and A/GO mismatches may be particularly important biological substrates for MutY. Proteolytic digestion of MutY using thermolysin was found to produce two relatively stable fragments of 25 and 12 kDa. The 25 kDa fragment begins at the N terminus of MutY and spans the region homologous with E. coli endonuclease III, a DNA glycosylase/AP lyase that repairs oxidatively damaged pyrimidines. The 12 kDa fragment, which consists of much of the rest of MutY, had no detectable activity. The purified 25 kDa fragment (M25) had nearly wild-type binding and cleavage activities with A/G-mismatched substrates. Binding to A/GO-mismatched DNA, however, was dramatically reduced in M25 compared to that in intact protein. Borohydride-dependent enzyme-DNA cross-linking, which is a hallmark of the reaction of several DNA glycosylases that possess concomitant AP lyase activity, was also substantially reduced when M25 was allowed to react with A/GO-mismatched DNA. The significant differences in M25 recognition and reactivity with A/G and A/GO mismatches suggest that the C-terminal region of MutY, a region with no homologous counterpart in E. coli endonuclease III, plays an important role in the repair of mismatched DNA arising from oxidation damage.

Kavli B et al.
Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase.
EMBO J. 1996; 15: 3442-7
Display abstract
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.

Gill RD, Cussac C, Souhami RL, Laval F
Increased resistance to N,N',N"-triethylenethiophosphoramide (thiotepa) in cells expressing the Escherichia coli formamidopyrimidine-DNA glycosylase.
Cancer Res. 1996; 56: 3721-4
Display abstract
Thiotepa (N,N',N'-triethylenethiophosphoramide) is an alkylating agent used in cancer chemotherapy. A reaction pathway by which thiotepa alkylates purified DNA involves hydrolysis to aziridine, which forms N7-aminoethyl guanine and aminoethyl adenine. These lesions are repaired in Escherichia coli by the formamidopyrimidine-DNA glycosylase (Fpg) protein. To determine whether such lesions are formed by thiotepa in mammalian cells, we have overexpressed the E. coli Fpg protein in Chinese hamster ovary cells. The transfected cells were more resistant to the lethal and mutagenic effects of thiotepa than the parental cells. The number of replication-blocking lesions formed by thiotepa, measured by quantitative PCR analysis, was lower in the transfected cells. These results show that expression of the Fpg protein increases the cell resistance to thiotepa and suggest that this compound produces ring-opened guanines, which are involved in its cytotoxic action.

Manuel RC, Czerwinski EW, Lloyd RS
Identification of the structural and functional domains of MutY, an Escherichia coli DNA mismatch repair enzyme.
J Biol Chem. 1996; 271: 16218-26
Display abstract
The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments. NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A.G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2'-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.

Engelward BP, Dreslin A, Christensen J, Huszar D, Kurahara C, Samson L
Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing.
EMBO J. 1996; 15: 945-52
Display abstract
In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.

Memisoglu A, Samson L
Cloning and characterization of a cDNA encoding a 3-methyladenine DNA glycosylase from the fission yeast Schizosaccharomyces pombe.
Gene. 1996; 177: 229-35
Display abstract
We have begun to develop the fission yeast, Schizosaccharomyces pombe, as a eukaryotic model for cellular defenses against alkylating agents. Here we describe the cloning and characterization of a cDNA, designated mag1, encoding a S. pombe 3-methyladenine (3MeA) DNA glycosylase. 3MeA DNA glycosylases in Escherichia coli are encoded by alkA and tag. S. pombe mag1 was cloned by its ability to reverse the alkylation-sensitive phenotype of an alkA tag E. coli double mutant. The expression of S. pombe mag1 in E. coli confers partial resistance to alkylating agents that produce methyl, ethyl and propyl lesions, and Mag1 production produces 3MeA DNA glycosylase activity. In contrast to the E. coli alkA and Saccharomyces cerevisiae MAG genes, expression of S. pombe mag1 was not appreciably induced by alkylating agents. The mag1 cDNA encodes a protein of 228 amino acids (aa) that shares similarity with 3MeA DNA glycosylases from E. coli (AlkA), Bacillus subtilis (BsAlkA) and S. cerevisiae (MAG). A consensus sequence of 9 aa common to these microbial 3MeA DNA glycosylases is discussed.

Matijasevic Z, Stering A, Niu TQ, Austin-Ritchie P, Ludlum DB
Release of sulfur mustard-modified DNA bases by Escherichia coli 3-methyladenine DNA glycosylase II.
Carcinogenesis. 1996; 17: 2249-52
Display abstract
The toxic effects of sulfur mustard have been attributed to DNA modification with the formation of 7-hydroxyethylthioethyl guanine, 3-hydroxyethylthioethyl adenine and the cross-link, di-(2-guanin-7-yl-ethyl)sulfide. To investigate the action of bacterial 3-methyladenine DNA glycosylase II (Gly II) on these adducts, calf thymus DNA was modified with [14C]sulfur mustard and used as a substrate for Gly II. Gly II releases both 3-hydroxyethylthioethyl adenine and 7-hydroxyethylthioethyl guanine from this substrate. In comparison with the activity of Gly II towards methylated DNA, 3-hydroxyethylthioethyl adenine is released somewhat more slowly than 3-methyladenine, while 7-hydroxyethylthioethyl guanine is released much more readily than 7-methylguanine. Glycosylase action may play a role in protecting cells from the toxic effects of sulfur mustard.

Kamada K, Horiuchi T, Ohsumi K, Shimamoto N, Morikawa K
Structure of a replication-terminator protein complexed with DNA.
Nature. 1996; 383: 598-603
Display abstract
The crystal structure of the Escherichia coli replication-terminator protein (Tus) bound to terminus-site (Ter) DNA has been determined at 2.7 A resolution. The Tus protein folds into a previously undescribed architecture divided into two domains by a central basic cleft. This cleft accommodates locally deformed B-form Ter DNA and makes extensive contacts with the major groove, mainly through two interdomain beta-strands. The unusual structural features of this complex may explain how the replication fork is halted in only one direction.

Lu AL, Yuen DS, Cillo J
Catalytic mechanism and DNA substrate recognition of Escherichia coli MutY protein.
J Biol Chem. 1996; 271: 24138-43
Display abstract
Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities. In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.

Cussac C, Laval F
Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene.
Nucleic Acids Res. 1996; 24: 1742-6
Display abstract
Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.

Eide L, Bjoras M, Pirovano M, Alseth I, Berdal KG, Seeberg E
Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.
Proc Natl Acad Sci U S A. 1996; 93: 10735-40
Display abstract
One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

Bjelland S, Seeberg E
Different efficiencies of the Tag and AlkA DNA glycosylases from Escherichia coli in the removal of 3-methyladenine from single-stranded DNA.
FEBS Lett. 1996; 397: 127-9
Display abstract
Escherichia coli possesses two different DNA repair glycosylases, Tag and AlkA, which have similar ability to remove the alkylation product 3-methyladenine from double-stranded DNA. In this study we show that these enzymes have quite different activities for the excision of 3-methyladenine from single-stranded DNA, AlkA being 10-20 times more efficient than Tag. We propose that AlkA and perhaps other glycosylases as well may have an important role in the excision of base damage from single-stranded regions transiently formed in DNA during transcription and replication.

Lu AL, Tsai-Wu JJ, Cillo J
DNA determinants and substrate specificities of Escherichia coli MutY.
J Biol Chem. 1995; 270: 23582-8
Display abstract
Potential DNA contacts involved in the specific interaction between the Escherichia coli MutY protein and a 40-mer oligonucleotide containing an A/G mismatch have been examined by alkylation interference techniques. Ethylation interference patterns suggest that more than five phosphates are involved in electrostatic interactions between MutY and DNA. Interestingly, MutY has more contacts on the G-strand than on the A-strand. Methylation at both the N-7 position of the mismatched G and the N-3 position of the mispaired A interfere with MutY binding. In addition to these mismatched bases, MutY also contacts purines on both sides of the mismatch. Binding and endonuclease activities of MutY were assayed with 20-mer oligonucleotides containing A/G, A/C, A/7,8-dihydro-8-oxo-guanine (A/GO), A/inosine (A/I), A/2-aminopurine (A/2AP), nebularine/G (N/G), inosine/G (I/G), 2AP/G, and 7-deaza-adenosine/G (Z/G) mispairs. The C-8 keto group of GO in A/GO contributes to a much tighter binding but weaker endonuclease activity than is seen with A/G. Because A/I is not specifically well recognized by MutY, the 2-amino group of G in A/G is essential for recognition. The C-6 keto group present in A/G but absent in A/2AP is also important for recognition. The 6-amino group of adenine appears not to be required for either binding or endonuclease activity because N/G is as good a substrate as A/G. The 2AP/G mispair is bound and cleaved weaker than is the A/G mispair. Binding and endonuclease activities are abolished when the N-7 group of A is replaced by C-7 as in the Z/G mispair. When a C-6 keto group is present as in the I/G pair, its binding by MutY is as good as for A/G, but no endonuclease activity is observed. Taken together, our data suggest that DNA sequences proximal to and specific functional groups of mismatched bases are necessary for recognition and catalysis by MutY protein.

Saparbaev M, Kleibl K, Laval J
Escherichia coli, Saccharomyces cerevisiae, rat and human 3-methyladenine DNA glycosylases repair 1,N6-ethenoadenine when present in DNA.
Nucleic Acids Res. 1995; 23: 3750-5
Display abstract
The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which generate various ethenobases in DNA. It has been reported that 1,N6-ethenoadenine (epsilon A) is excised by a DNA glycosylase present in human cell extracts, whereas protein extracts from Escherichia coli and yeast were devoid of such an activity. We confirm that the human 3-methyladenine-DNA glycosylase (ANPG protein) excises epsilon A residues. This finding was extended to the rat (ADPG protein). We show, at variance with the previous report, that pure E.coli 3-methyladenine-DNA glycosylase II (AlkA protein) as well as its yeast counterpart, the MAG protein, excise epsilon A from double stranded oligodeoxynucleotides that contain a single epsilon A. Both enzymes act as DNA glycosylases. The full length and the truncated human (ANPG 70 and 40 proteins, respectively) and the rat (ADPG protein) 3-methyladenine-DNA glycosylases activities towards epsilon A are 2-3 orders of magnitude more efficient than the E.coli or yeast enzyme for the removal of epsilon A. The Km of the various proteins were measured. They are 24, 200 and 800 nM for the ANPG, MAG and AlkA proteins respectively. These three proteins efficiently cleave duplex oligonucleotides containing epsilon A positioned opposite T, G, C or epsilon A. However the MAG protein excises A opposite cytosine much faster than opposite thymine, guanine or adenine.

Mol CD et al.
Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA.
Cell. 1995; 82: 701-8
Display abstract
Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.

Bjoras M, Klungland A, Johansen RF, Seeberg E
Purification and properties of the alkylation repair DNA glycosylase encoded the MAG gene from Saccharomyces cerevisiae.
Biochemistry. 1995; 34: 4577-82
Display abstract
The MAG gene of Saccharomyces cerevisiae encodes an alkylation repair DNA glycosylase whose sequence is homologous to the AlkA DNA glycosylase from Escherichia coli. To investigate the biochemical properties of MAG in comparison to AlkA, MAG was expressed in E. coli and purified to electrophoretic homogeneity. N-Terminal sequencing of the purified protein identified the translational start site which corresponded to that predicted previously from the nucleotide sequence. Polyclonal antibodies raised against MAG inhibited the enzymatic activity of MAG, but not that of AlkA, and vice versa, implying that the structures of the active site regions of these enzymes are antigenic, but sufficiently different to have different epitopes. Kinetic analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea and [3H]dimethyl sulfate showed that MAG was as effective as AlkA in removing 3-methyladenine, 7-methylguanine, and 7-methyladenine. However, the purified MAG enzyme did not catalyze the excision of O2-methylthymine, which is a major substrate for AlkA. Furthermore, 3-methylguanine was excised 20-40 times more slowly by MAG than by AlkA. The kinetics of 3-methylguanine excision by MAG were found to be similar to the low rate of 3-methylguanine excision catalyzed by 3-methyladenine DNA glycosylase I (Tag) of E. coli. Expression of MAG in alkA mutant cells did not effectively restore alkylation resistance of the mutant as did AlkA itself. It thus appears that MAG is a less versatile enzyme than AlkA in spite of the sequence relationship and may have a similar function in yeast as the nonhomologous Tag enzyme in E. coli.

Xing D, Dorr R, Cunningham RP, Scholes CP
Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching.
Biochemistry. 1995; 34: 2537-44
Display abstract
We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has two tryptophans, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., & Cunningham, R. P. (1992) Science 258, 434-440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher. Significant (approximately 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT). The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS)

Park HW, Kim ST, Sancar A, Deisenhofer J
Crystal structure of DNA photolyase from Escherichia coli.
Science. 1995; 268: 1866-72
Display abstract
Photolyase repairs ultraviolet (UV) damage to DNA by splitting the cyclobutane ring of the major UV photoproduct, the cis, syn-cyclobutane pyrimidine dimer (Pyr <> Pyr). The reaction is initiated by blue light and proceeds through long-range energy transfer, single electron transfer, and enzyme catalysis by a radical mechanism. The three-dimensional crystallographic structure of DNA photolyase from Escherichia coli is presented and the atomic model was refined to an R value of 0.172 at 2.3 A resolution. The polypeptide chain of 471 amino acids is folded into an amino-terminal alpha/beta domain resembling dinucleotide binding domains and a carboxyl-terminal helical domain; a loop of 72 residues connects the domains. The light-harvesting cofactor 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) binds in a cleft between the two domains. Energy transfer from MTHF to the catalytic cofactor flavin adenine dinucleotide (FAD) occurs over a distance of 16.8 A. The FAD adopts a U-shaped conformation between two helix clusters in the center of the helical domain and is accessible through a hole in the surface of this domain. Dimensions and polarity of the hole match those of a Pyr <> Pyr dinucleotide, suggesting that the Pyr <> Pyr "flips out" of the helix to fit into this hole, and that electron transfer between the flavin and the Pyr <> Pyr occurs over van der Waals contact distance.

Pearl LH, Savva R
DNA repair in three dimensions.
Trends Biochem Sci. 1995; 20: 421-6

Chaudhry MA, Weinfeld M
The action of Escherichia coli endonuclease III on multiply damaged sites in DNA.
J Mol Biol. 1995; 249: 914-22
Display abstract
Energy deposition by ionizing radiation can lead to the formation of clustered DNA damage, i.e. more than one lesion situated within a helical turn of DNA. Among the postulated lesions are those characterized by damaged bases and abasic sites on opposite strands. Enzymatic removal of such lesions may inadvertently lead to the formation of double-strand breaks. To test this hypothesis, we have constructed model substrates containing damaged bases (5,6-dihydrothymine) or abasic sites set one, three, five and seven bases apart on opposite strands, and examined the reactivity of Escherichia coli endonuclease III towards these substrates. Endonuclease III demonstrates two activities; as a glycosylase that removes saturated pyrimidine bases, such as dihydrothymine, and as an AP lyase that cleaves DNA strands at abasic sites. Analysis of endonuclease III-treated dihydrothymidine containing plasmid DNA by agarose gel electrophoresis indicated that the enzyme generated only single-strand breaks when the base damage was set one and three base-pairs apart, and only slowly introduced double-strand breaks in the other substrates. Endonuclease III treatment of the abasic site-containing DNA, however, readily yielded double-strand breaks. Taken together, these results indicate that the glycosylase activity of the enzyme, but not the AP lyase activity, is inhibited by the presence of a closely positioned break in the opposite strand.

Mol CD et al.
Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.
Cell. 1995; 80: 869-78
Display abstract
Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

Visse R, van Gool AJ, Moolenaar GF, de Ruijter M, van de Putte P
The actual incision determines the efficiency of repair of cisplatin-damaged DNA by the Escherichia coli UvrABC endonuclease.
Biochemistry. 1994; 33: 1804-11
Display abstract
The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.

Saparbaev M, Laval J
Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.
Proc Natl Acad Sci U S A. 1994; 91: 5873-7
Display abstract
The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.

Dosanjh MK, Roy R, Mitra S, Singer B
1,N6-ethenoadenine is preferred over 3-methyladenine as substrate by a cloned human N-methylpurine-DNA glycosylase (3-methyladenine-DNA glycosylase).
Biochemistry. 1994; 33: 1624-8
Display abstract
A lethal DNA adduct induced by methylating agents, 3-methyladenine (m3A), is removed by both the constitutive (Tag) and inducible (AlkA) bacterial m3A-DNA glycosylases. The human 3-methyladenine-DNA glycosylase also releases m3A as well as other methylated bases. The rate of release of m3A from alkylated DNA by the purified or recombinant human m3A glycosylase is much higher than that of the other methylated bases. We now find that a partially purified recombinant human m3A-DNA glycosylase, expressed in Escherichia coli, releases at least 10-fold more 1,N6-ethenoadenine (epsilon A) than m3A from DNA. epsilon A is completely unrelated to m3A since it is a heterocyclic adduct produced by the carcinogen vinyl chloride. The rates of release of epsilon A and m3A were both dependent on protein concentration and time. The differential release of epsilon A and m3A occurs regardless of whether DNA containing each adduct is assayed separately or is assayed in a mixed substrate containing both DNAs. This result raises the question of what structural features are involved in recognition and excision by the human m3A-DNA glycosylase and what may be its primary substrate.

Eftedal I, Volden G, Krokan HE
Excision of uracil from double-stranded DNA by uracil-DNA glycosylase is sequence specific.
Ann N Y Acad Sci. 1994; 726: 312-4

Kumar NV, Varshney U
Inefficient excision of uracil from loop regions of DNA oligomers by E. coli uracil DNA glycosylase.
Nucleic Acids Res. 1994; 22: 3737-41
Display abstract
Kinetic parameters for uracil DNA glycosylase (E. coli)-catalysed excision of uracil from DNA oligomers containing dUMP in different structural contexts were determined. Our results show that single-stranded oligonucleotides (unstructured) are used as somewhat better substrates than the double-stranded oligonucleotides. This is mainly because of the favourable Vmax value of the enzyme for single-stranded substrates. More interestingly, however, we found that uracil release from loop regions of DNA hairpins is extremely inefficient. The poor efficiency with which uracil is excised from loop regions is a result of both increased Km and lowered Vmax values. This observation may have significant implications in uracil DNA glycosylase-directed repair of DNA segments that can be extruded as hairpins. In addition, these studies are useful in designing oligonucleotides for various applications in DNA research where the use of uracil DNA glycosylase is sought.

Mroczkowska MM, Kolasa IK, Kusmierek JT
Chloroacetaldehyde-induced mutagenesis in Escherichia coli: specificity of mutations and modulation by induction of the adaptive response to alkylating agents.
Mutagenesis. 1993; 8: 341-8
Display abstract
The mutational specificity of chloroacetaldehyde (CAA), one of the metabolites of the human carcinogen vinyl chloride (VC), has been determined through the examination of Arg+ revertants in Escherichia coli AB2497 (Arg-) and identification of their tRNA suppressors. The predominant mutations were GC-->AT transitions (65%) followed by AT-->TA transversions (12.5%). The observed mutational specificity of CAA is very similar to the reported specificity of the other VC metabolite, chloroethylene oxide. The induction of the adaptive response to alkylating agents significantly decreased the frequency of CAA-induced Rifr and Arg+ mutants in E. coli AB2497 and increased the cell survival. Likewise, the adaptation of bacterial cells decreased the frequency of GC-->AT transitions in CAA-treated M13glyU phage transformed to E. coli JC15419 and increased the phage survival. Experiments with strain MS23, which is an alkA mutant deficient in 3-methyladenine-DNA glycosylase II, and with MS23 harboring the pYN1000 plasmid carrying the alkA+ gene, have shown that induction of this repair enzyme is responsible for reduction of the level of CAA-induced mutations. The role of N2,3-ethenoguanine, among the other etheno-adducts, in CAA-induced mutagenesis and as a target for repair in 3-methyladenine-DNA glycosylase II proficient bacterial cells is discussed.

Engelward BP, Boosalis MS, Chen BJ, Deng Z, Siciliano MJ, Samson LD
Cloning and characterization of a mouse 3-methyladenine/7-methyl-guanine/3-methylguanine DNA glycosylase cDNA whose gene maps to chromosome 11.
Carcinogenesis. 1993; 14: 175-81
Display abstract
In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions by DNA glycosylases prevents alkylation induced cell death. We described previously the isolation of a human 3MeA DNA glycosylase (AAG) cDNA that maps to chromosome 16 and hybridizes to specific genomic DNA fragments from a number of mammals, including mouse. As a first step in the generation of a 3MeA DNA glycosylase deficient mouse by homologous replacement in embryonic stem cells, we have cloned the mouse 3MeA DNA glycosylase cDNA. The cloned 1095 base pair cDNA contains a complete 333 amino acid open reading frame that predicts a 36.5 kDa protein and hybridizes to a 1.5 kb mRNA transcript. Mouse 3MeA DNA glycosylase (Aag) transcript levels vary by up to 21 fold among tissues, being highest in the testes and lowest in the heart. The Aag cDNA encodes a glycosylase able to release 3MeA, 7-methylguanine (7MeG) and 3-methylguanine (3MeG) from alkylated DNA. The expression of Aag in E. coli provides substantial resistance against killing by methylating agents, but, unlike its E. coli counterparts, the Aag glycosylase fails to protect against killing by ethylating and propylating agents. A 232 amino acid stretch of the predicted mouse protein shares extensive amino acid identity with rat (93%) and human (83%) 3MeA DNA glycosylases and we observe that all three mammalian glycosylases have a bipartite nuclear localization signal. The Aag gene maps to mouse chromosome 11, suggesting a segment of conserved synteny between mouse chromosome 11 and human chromosome 16, which bears the human 3MeA DNA glycosylase gene. Cloning the mouse 3MeA DNA glycosylase cDNA is a step toward understanding the role of this DNA repair enzyme in mammals.

Higley M, Lloyd RS
Processivity of uracil DNA glycosylase.
Mutat Res. 1993; 294: 109-16
Display abstract
The purpose of this study was to determine the mechanism by which uracil DNA glycosylase locates uracil residues within double-stranded DNA. Using reaction conditions that contained low salt concentrations, the addition of uracil DNA glycosylase to plasmid DNAs containing multiple, randomly incorporated uracils resulted in the accumulation of form III DNA while unreacted form I DNA was still present. These data suggested that the enzyme utilizes a one-dimensional DNA-scanning mechanism such that this linear DNA arose by the accumulation of many single-strand breaks within the plasmid prior to enzyme dissociation. Reactions containing higher concentrations of uracil DNA glycosylase revealed a further accumulation of form III DNA after all form I DNA had been lost. These results suggested a partial (1.5-2 kb) enzyme processivity since the enzyme does not incise at all uracil bases on the DNA molecule prior to dissociation from that DNA. Since DNA scanning is regulated by electrostatic interactions, the processivity of the enzyme was evaluated through kinetic analyses of incision at various salt concentrations. At NaCl concentrations (< 50 mM), a significant amount of form III DNA accumulated while there were still unreacted form I DNAs present. In contrast, the accumulation of form III DNA was delayed at higher salt concentrations and the overall accumulation of form III DNA was less than that monitored at lower salt concentrations. DNAs were also analyzed by denaturing agarose gel electrophoresis in order to measure the average distance between strand breaks. Southern hybridizations showed a greater accumulation of breaks in DNAs that were reacted with the uracil DNA glycosylase at the lower salt concentrations, confirming a partial processivity for the enzyme.

Bessho T et al.
Repair of 8-hydroxyguanine in DNA by mammalian N-methylpurine-DNA glycosylase.
Proc Natl Acad Sci U S A. 1993; 90: 8901-4
Display abstract
8-Hydroxyguanine is one of the major base lesions implicated in mutagenesis induced by ionizing radiation and radiomimetic agents. This lesion appears to be repaired by human cells via multiple pathways including the one that involves a base glycosylase. Mouse N-methylpurine-DNA glycosylase, responsible for the removal of N-alkylpurines in DNA that are induced by simple monofunctional alkylating agents, also releases 8-hydroxyguanine from DNA in vitro and in vivo in Escherichia coli. The human N-methylpurine-DNA glycosylase, with a lower preference for N-alkylguanine than the mouse protein, removes the oxidized base less efficiently than the mouse protein. The recombinant mammalian glycosylases can rescue E. coli lacking MutM (Fpg) protein, the DNA glycosylase that is primarily responsible for removing 8-hydroxyguanine from the bacterial DNA.

Bjelland S, Bjoras M, Seeberg E
Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli.
Nucleic Acids Res. 1993; 21: 2045-9
Display abstract
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.

Habraken Y, Laval F
Increased resistance of the Chinese hamster mutant irs1 cells to monofunctional alkylating agents by transfection of the E. coli or mammalian N3-methyladenine-DNA-glycosylase genes.
Mutat Res. 1993; 293: 187-95
Display abstract
Irs1 cells are mutants of the Chinese hamster cell line V79-4, and exhibit cross-sensitivity to various DNA-damaging agents, especially to the alkylating compounds methyl methanesulfonate and ethyl methanesulfonate. To test whether this sensitivity was due to the persistence of alkylated residues in DNA, we have transfected irs1 cells with the pMSG expression vector containing two coding sequences for enzymes of different origin, either the E. coli AlkA gene, coding for 3-methyladenine-DNA-glycosylase II, or rat APDG cDNA, encoding alkylpurine-DNA-glycosylase. The two coding sequences for the repair enzymes were ligated in the pMSG vector, under the control of the MMTV-LTR promoter, which is responsive to glucocorticoid regulation. The presence of the AlkA gene or of the APDG cDNA in the transfected cells was detected by Southern blot analysis and the transcription of these foreign sequences was checked by Northern hybridization of the cellular RNA. The transfected irs1 cells treated with [3H]dimethylsulfate removed the 3-methyladenine residues more efficiently from their DNA than the control cells. Irs1 cells harboring the AlkA or the APDG gene become about 2- and 3-fold more resistant to the toxic effect of methyl methanesulfonate, respectively. However, a 3-fold resistance to ethyl methanesulfonate was only observed in irs1 cells harboring the mammalian APDG cDNA.

Morohoshi F, Hayashi K, Munkata N
Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon.
J Bacteriol. 1993; 175: 6010-7
Display abstract
In Bacillus subtilis, the adaptive response to DNA alkylation depends on the ada operon, which consists of the adaA and adaB genes, which encode methylphosphotriester DNA methyltransferase (AdaA protein) and O6-methylguanine DNA methyltransferase (AdaB protein), respectively. A structural gene (alkA) that encodes 3-methyladenine DNA glycosylase was found upstream of the ada operon, but in the opposite orientation. This cluster of genes was mapped at about 235 kb from the SfiI recognition site near the origin of replication in the physical map of the B. subtilis chromosome. Disruption of the alkA gene sensitized cells to N-propyl-N'-nitro-N-nitrosoguanidine, while its overproduction rendered cells highly resistant to N-propyl-N'-nitro-N-nitrosoguanidine, indicating that lethal DNA damage produced by bulky alkylating agents was effectively counteracted by AlkA glycosylase. Transcription of the alkA gene was induced by treating adaA+ cells with methylating agents concurrent with transcription of the ada operon. This was accomplished by using methylated AdaA protein bound to a 30-bp segment in the middle of the 100-bp sequence between the transcriptional start sites of the alkA gene and ada operon. Thus, in this organism, the adaptive response to DNA alkylation is achieved by autologous activation of a divergent regulon composed of the genes for a DNA glycosylase and two species of DNA alkyltransferase.

Mauro DJ, De Riel JK, Tallarida RJ, Sirover MA
Mechanisms of excision of 5-fluorouracil by uracil DNA glycosylase in normal human cells.
Mol Pharmacol. 1993; 43: 854-7
Display abstract
Recent evidence indicates that 5-fluorouracil (5-FlUra) is incorporated into DNA and is removed by the DNA repair enzyme uracil DNA glycosylase. Synthetic oligonucleotides containing either a single uracil or 5-FlUra residue were constructed to examine the mechanisms by which human cells remove 5-FlUra from DNA. The human uracil DNA glycosylase excised uracil in a manner similar to that observed for the bacterial enzyme. In contrast, a significant difference was observed in their abilities to remove 5-FlUra. In particular, both the bacterial and normal human enzymes displayed 13-17-fold increases in their apparent Km values but the apparent Vmax values remained virtually constant. These results demonstrate that normal human cells possess a defined capacity to remove 5-FlUra incorporated into DNA. However, specific kinetic differences may exist that affect their capacity to remove 5-FlUra formed in DNA after treatment with this cancer chemotherapeutic agent.

Matijasevic Z, Sekiguchi M, Ludlum DB
Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.
Proc Natl Acad Sci U S A. 1992; 89: 9331-4
Display abstract
The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14C]guanine-containing DNA substrate by nick-translating DNA with [14C]dGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cell might protect them from exposure to metabolites of vinyl chloride.

Singer B et al.
Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.
Proc Natl Acad Sci U S A. 1992; 89: 9386-90
Display abstract
We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.

Habraken Y, Carter CA, Sekiguchi M, Ludlum DB
Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.
Carcinogenesis. 1991; 12: 1971-3
Display abstract
3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.

Riazuddin S, Athar A, Ahmed Z, Lali SM, Sohail A
DNA glycosylase enzymes induced during chemical adaptation of M. luteus.
Nucleic Acids Res. 1987; 15: 6607-24
Display abstract
Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively.

Helland DE, Male R, Haukanes BI, Olsen L, Haugan I, Kleppe K
Properties and mechanism of action of eukaryotic 3-methyladenine-DNA glycosylases.
J Cell Sci Suppl. 1987; 6: 139-46
Display abstract
3-Methyladenine-DNA glycosylase activities have been identified in all eukaryotic cell systems studied. Some of the results from these studies are reviewed here. The enzymes possess molecular weights between 24 X 10(3) and 34 X 10(3), they have a broad pH optimum at approximately pH 8, require double-stranded DNA and act in the absence of any cofactors. The enzyme can excise several different methylated bases from DNA such as 3-methyladenine, 7-methylguanine and 3-methylguanine. The specific activity of this DNA glycosylase in mouse L-cells was found to be a function of the proliferative state of the cell. In vitro quantification of this DNA repair activity in synchronized mouse L-cells suggests that it is regulated within a defined temporal sequence prior to the onset of DNA replication. Using DNA fragments of defined sequences it was observed that the efficiency of removal of the methylated bases is sequence-dependent.

Male R, Haukanes BI, Helland DE, Kleppe K
Substrate specificity of 3-methyladenine-DNA glycosylase from calf thymus.
Eur J Biochem. 1987; 165: 13-9
Display abstract
3-Methyladenine-DNA glycosylase from calf thymus recognizes both 3-methyladenine (3-mAde), 7-methylguanine (7-mGua) and 3-methylguanine (3-mGua) residues in calf thymus DNA; the rate of release of 3-mAde is approximately eightfold higher than that for 7-mGua. The best DNA polymer substrates appeared to be those having an A-type helical conformation such as d(A-T)n and d(G-C)n. The Km values for release of 3-mAde and 7-mGua were approximately the same for the above mentioned two substrates whereas the Vmax for excision of 3-mAde was threefold higher than that of 7-mGua. The rate of hydrolysis of 7-mGua residues in d(G-C)n was similar to that found for the excision of 3-mAde in calf thymus DNA. The polymer d(G)n X d(C)m, which possesses a B-type helical conformation, was a poor substrate and the rate of excision here was approximately the same as with calf thymus DNA having the B-type structure. Polyamines greatly influenced the activity and at low concentrations a 50-100% increase in the release of 7-mGua, but not 3-mAde, was observed. With higher concentrations the rate of excision of both bases decreased sharply. The sequence specificity of the DNA glycosylase on naturally occurring DNA was studied using methylated DNA fragments from the plasmid pUC18. The results revealed that some 3-mAde as well as 7-mGua residues were seldom attacked. These 3-mAde residues were positioned either 5' to another Ade residue or in a stretch of pyrimidines, and the 7-mGua residue 3' to another Gua residue. The 3-mAde residue most frequently recognized was situated 3' to another Ade residue, and in the case of 7-mGua it was the central Gua residue in the sequence -G-G-G-.

Kaasen I, Evensen G, Seeberg E
Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance.
J Bacteriol. 1986; 168: 642-7
Display abstract
We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II. The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes. High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA. Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair.

Evensen G
Induction of 3-methyladenine DNA glycosylase II is recA+-independent.
Mutat Res. 1985; 146: 143-7
Display abstract
The recA1 mutation was transduced into the tag-2 mutant of E. coli, thus making a strain deficient in the induction of SOS repair as well as in the constitutive repair of 3-alkylated adenines in DNA. The double mutant recA tag is more sensitive to methyl methanesulfonate exposure than either single mutant, indicating that recA and tag mutations block different pathways in repair of alkylation damage. The double mutant is more deficient in host cell reactivation of alkylated phages than the tag single mutant. However, alkylation induction of the double mutant with N-methyl-N'-nitro-N-nitrosoguanidine resulted in killing adaptation of the cells to methyl methanesulfonate and restored the host cell reactivation capacity for alkylated lambda phage to wild-type levels. These adaptive responses can be ascribed to the induction of 3-methyladenine DNA glycosylase II which is shown by enzyme analysis to proceed normally in the recA mutant background. The results imply that the induction of the alkA gene encoding 3-methyladenine DNA glycosylase II is independent of SOS induction.

Nakabeppu Y, Kondo H, Sekiguchi M
Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II.
J Biol Chem. 1984; 259: 13723-9
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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.

Clarke ND, Kvaal M, Seeberg E
Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II.
Mol Gen Genet. 1984; 197: 368-72
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We have constructed two recombinant plasmids which harbour functions involved in DNA repair of alkylation damage in Escherichia coli. One plasmid carries the tag+ gene encoding 3-methyladenine DNA glycosylase I while the other carries alkA+ encoding 3-methyladenine DNA glycosylase II. The plasmids were isolated from plasmid stocks carrying total cellular DNA by selection for their ability to complement the methylmethanesulphonate(MMS)-sensitive phenotype of an E. coli mutant (tag ada) deficient in both 3-methyladenine DNA glycosylases I and II. Both plasmids increase the plating efficiency of such a mutant on methylmethanesulphonate plates by a factor of more than 10(5). The tag gene is located on a 6 (kbp) HindIII fragment, and the presence of the tag plasmid in the cells results in 15-fold overproduction of 3-methyladenine DNA glycosylase I. The other plasmid restores 3-methyladenine DNA glycosylase II deficiency in alkA mutant cells, and results in 3-fold overproduction of this enzyme after alkylation induction. The induction is ada+-dependent and we conclude that this plasmid contains the structural gene for 3-methyladenine DNA glycosylase II, including its control region responding to alkylation induction. However, the plasmid does not complement fully the MMS-sensitive phenotype of alkA mutants which suggests that the plasmid may not include the entire alkA operon.

Ahmmed Z, Laval J
Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methylthymine-DNA methyltransferase and a O2-methylthymine DNA glycosylase.
Biochem Biophys Res Commun. 1984; 120: 1-8
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Alkylation of poly(dT) by N-[methyl-3H] (N-nitrosomethylurea) and subsequent annealing with poly(dA) yield a substrate containing O2 and O4-methylthymidine, 3-methylthymidine and phosphotriesters. In an in vitro assay using this substrate, cell extracts from Escherichia coli catalyse i) the transfer of the O4-methyl present in O4 methylthymidine to a protein which becomes alkylated; ii) the release of O2-methylthymine by a glycosylase activity. The two DNA repair activities described above appear to be involved in the adaptive response.

Price JA, Heller E, Goldthwait DA
The release of 3-methyladenine from nucleosomal DNA by a 3-methyladenine DNA glycosylase.
Carcinogenesis. 1983; 4: 145-52
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Nucleosomes from chicken erythrocytes, with DNA containing an average of 144 base pairs, were alkylated with [3H]methylnitrosourea. The level of alkylation of the nucleosomal DNA was 48% of that of free DNA. The histones had approximately one tenth the radioactivity of the DNA. There was no statistically significant difference between alkylation of nucleosome bases in the major vs. minor groove. When the first 50 residues of the alkylated nucleosomal DNA were examined on sequencing gels, the 7-methylguanine and 3-methyladenine (3-MeA) residues were distributed randomly. The 3-MeA DNA glycosylase I of E. coli was used to measure the release of 3-MeA from nucleosomal DNA. Incubation at 37 degrees C resulted in a release which reached a plateau of approximately 33% of the 3-MeA groups of the nucleosomal DNA. A partially purified 3-MeA DNA glycosylase from rat liver gave similar results. The limited enzymatic release is most likely due to steric hindrance of the enzyme by the DNA-histone interactions on the surface of the core particle. An alteration of nucleosomal conformation has been suggested as an explanation for repair of nucleosomal DNA. Two model systems have been examined. The addition of ethidium bromide to alkylated nucleosomes increased the enzymatic release of 3-MeA to approximately 75% and altered the electron microscopic appearance. The chemical alkylation of nucleosomes also increased the enzymatic release of 3-MeA as well as decreased the sedimentation coefficient. All of these experiments indicate a limited availability of 3-MeA residues to the glycosylase and suggest that some conformational change must occur in vivo for complete repair.

Karran P, Hjelmgren T, Lindahl T
Induction of a DNA glycosylase for N-methylated purines is part of the adaptive response to alkylating agents.
Nature. 1982; 296: 770-3

Mamet-Bratley MD, Karska-Wysocki B
Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage.
Biochim Biophys Acta. 1982; 698: 29-34
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Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204-213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5-10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.

Gallagher PE, Brent TP
Human 3-methyladenine-DNA glycosylase: demonstration of a stimulatory factor.
Biochem Biophys Res Commun. 1981; 101: 956-62