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Nucleic Acids Research, Vol. 18, No. 1 © 1990 Oxford University Press Active site amino acid sequence of the bovine O 6-methylguanine-DNA methyltransferase Bjorn Rydberg, Janet Hall* and Peter Karran Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK Received October 2, 1989; Revised and Accepted November 10, 1989 ABSTRACT An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins. INTRODUCTION O6-Methylguanine (m6-Gua) is one of the major products of the reaction of a number of methylating agents with DNA and is responsible for the potent mutagenicity of carcinogens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the metabolically activated form of dimethylnitrosamine (1). The spectrum of mutations induced by this group of methylating agents is dominated by GC to A T transition mutations (2,3) which is consistent with the observed propensity of m6-Gua to direct the incorporation of thymine into DNA in vitro (4). In addition to its well-documented mutagenicity, m6-Gua also contributes to other biological effects of alkylating agents. A number of human and rodent cell lines are unable to remove m6-Gua from their DNA. These cell lines are designated Mex~ (or Mer~) and are hypersensitive to the cytotoxic, clastogenic and mutagenic action of methylating agents (5,6). The hypersensitivity of cells which do not remove m6-Gua can be completely reversed by expression of a transfected E. coli ada+ gene encoding an m6-Gua repair function (7-9). Thus, m6-Gua is strongly implicated not only in the mutagenic, but also in the cytotoxic and clastogenic action of agents such as MNNG towards mammalian cells. In many bacteria (including E. coli (10), M. luteus (11), and B. subtilis (12)) the first line of defence against the biological effects of this methylated base is repair by specific DNA methyltransferases which demethylate the modified purine in situ but are without effect on other chemically-induced or naturally occurring methylated bases. The bacterial methyltransferases include the inducible Ada protein, a dual function methyltransferase comprising two domains which act separately to demethylate m6-Gua or methylphosphotriesters (10), and the constitutively expressed Ogt proteins off. coli (13) and the Datl (14) protein of B. subtilis. A common feature of these bacterial methyltransferases is transfer of a methyl group from the modified purine base onto a particular receptor cysteine residue within the methyltransferase molecule itself; the transfer being accompanied by an irreversible inactivation of the methyltransferase function. m6-Gua-DNA methyltransferase activities have been partially purified from several mammalian sources (15-18) and preliminary characterisation has indicated that they share a number of features with their bacterial counterparts. In particular, the automethyltransfer mode of repair has apparently been conserved and mammalian cells from a variety of sources (including human) are able to demethylate m6-Gua both in vivo and in cell-free extracts. In all cases, removal of methyl groups from m6-Gua in DNA is accompanied by a stoichiometric production of S-methylcysteine in a protease-sensitive form indicating that a cysteine residue serves as acceptor. Despite considerable efforts in a number of laboratories, mammalian methyltransferases have proved refractory to high yield purification and this has hampered further clarification of the mechanism of action of this important DNA repair enzyme. These difficulties have been partly due to excessive losses of the partially purified enzyme during the final stages of purification. Here we report the isolation and amino acid sequence of a peptide comprising the active site of the bovine enzyme. The derived sequence demonstrates a remarkable homology to the m6-GuaDNA methyltransferase active site of the E. coli Ada protein and the putative active site sequences of the E. coli Ogt and the B. subtilis Datl proteins. MATERIALS AND METHODS Materials Trypsin (Sequencing Grade) was obtained from Boehringer Mannheim. Sephadex G25 Superfine and the MonoS FPLC cation exchange column were obtained from Pharmacia and Ultrogel AcA54 from LKB. DE52 ion exchange cellulose and * Present address: International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, France 17 18 Nucleic Acids Research phosphocellulose PI 1 (Whatman) were prepared according to the manufacturers instructions. Single-stranded DNA cellulose was obtained from Sigma. Synthetic Oligonucleotides The 21mer oligonucleotide 5'-TGATCAGTAC(m6-G)CATGACTAGT-3' was synthesised by the phosphoramidite method on an Applied Biosystems Model 380B DNA synthesiser and further purified by HPLC. For use as a substrate for the methyltransferase, it was annealed to the oligomer 5'-ACTAGTCATGCGTACTGATCA-3' at a concentration of 2/iM in 0.1 M NaCl, lOmM TrisHCl pH 7.5, lmM EDTA at 37°C for 60min. m'-Gua-DNA Methyltransferase Assay [3H]-methylated M. luteus DNA was prepared using [3H]-Nmethyl-N-nitrosourea (MNU) (Amersham International, 24Ci/mmole) and partially depurinated as described (19). Assays were carried out in: 70mM Hepes KOH, pH 7.8/ lOmM dithiothreitol/ lmM EDTA. To monitor the purification procedure, 0.1— 2/il of each column fraction was incubated in 100/il reaction buffer containing [3H] substrate (lOOOcpm) for 20min at 37 °C. Following a digestion with proteinase K (125^g/120min) at 37°C, nucleic acids were precipitated with ethanol and the [3H] radioactivity in the supernatant was determined by scintillation counting. When appropriate, the removal of m6-Gua from the DNA was also monitored by published procedures (18). Partial Purification of m6-Gua-DNA Methyltransferase from Calf Thymus The procedure is based on that reported by Hall and Karran (18). All operations were performed at 0 - 4 ° C . 1.8Kg calf thymus freshly obtained from the slaughterhouse, was homogenised in 4 1 extraction buffer (0.1M NaCl, 50mM TrisHCl pH 7.5, lmM EDTA, 0.1% /3-mercaptoethanol, 0.1% Triton X-100, lmM phenylmethylsulphonyl fluoride, and 0.5/ig/ml each: leupeptin, pepstatin and chymostatin) using a Waring blendor at maximum setting for 2 x30sec. Extraction was for 45min at 0°C. Tissue debris was then removed by centrifugation at 3200 Xg for 30 min. To 41 supernatant was added a thick slurry of 1.21 DE52 in buffer A (50mM NaCl, 20mM Tris HC1 pH 7.5, lmM K 2 HPO 4 , lmM EDTA, lmM dithiothreitol, 0.1% |3mercaptoethanol). The mixture was stirred for 30min and the DE52 allowed to settle. The supernatant was decanted and the DE52 was then washed with 41 buffer A. To the two combined supernatants (81) was added 21 phosphocellulose PI 1 as a thick slurry in buffer A. The mixture was stirred for lhr, the PI 1 was then allowed to settle and the supernatant was discarded. The PI 1 was washed twice with 81 buffer A and then poured into a column (35cmx8.5cm diam.) which was eluted with buffer A containing 0.5M NaCl at a flow rate of 200ml/h. Fractions containing methyltransferase activity were combined (500ml) and dialysed against 201 buffer B (lmM potassium phosphate pH 7.5, lmM EDTA, 0.1% /3-mercaptoethanol, 10% glycerol) for 18h. The dialysed sample was clarified by centrifugation at 15000xg for 30 min and loaded on a single-stranded DNA cellulose column (12cm x4cm diam.) equilibrated with buffer B. The column was washed with 3 column volumes of buffer B and then eluted successively with with 2 column volumes each of buffer B containing 0.1M NaCl, 0.3M NaCl and 1M NaCl at a flow rate of 40ml/h. The activity was eluted with 1M NaCl, although the enzyme has typically been eluted with 0.3M NaCl with other batches of DNA cellulose. The pooled active fractions (25ml) were loaded onto a column of Ultrogel AcA54 (120 cmx2.5cm diam.) equilibrated with buffer C (0.1M NaCl, 15mM potassium phosphate pH 7.4, lmM EDTA, 0.1 % j3-mercaptoethanol, 10% glycerol). At this NaCl concentration, the active fractions were eluted at 1.3-1.5 times the void volume of the column (Vo), which is earlier than previously reported (1.8x Vo) when buffer C containing 0.5M NaCl was used. This altered elution behaviour indicates possible aggregation of the methyltransferase or interaction with other proteins at the lower salt concentration. The active fractions were pooled and concentrated using an Amicon ultrafiltration cell equipped with a Diaflo YM10 membrane. Total yield was about 800 pmole active enzyme (0.5% recovery) with a specific activity of 300 units/mg protein (500-fold purification). Incubation of the enzyme with [3H]-labelled substrate followed by SDS-Page electrophoresis and fluorography showed a major radioactive product of about 24kD (and a minor labelled species at about 27kD, probably resulting from incomplete denaturation of the labelled protein) in good agreement with previous estimates of the molecular mass of the protein (17). Labelling the Enzyme and Binding to a Solid Support An estimated total of 600pmole partly purified enzyme in 6ml buffer C was dialysed into reaction buffer by ultrafiltration using a Diaflo YM10 filter and the volume adjusted to 20ml. A 2ml aliquot of this preparation was incubated for 30min at 37 °C with 106 cpm of [3H]-labelled DNA substrate in a glass test-tube, 18ml was similarly incubated with lnmole m6-Gua-containing oligonucleotide. The two reaction mixtures were then combined. 2.4g siliconized glass wool, prepared by immersion in Repelcote (BDH) followed by several washes in distilled H2O, was then added and the methylated enzyme was allowed to bind to the glass wool by incubation for a further 30 min at 37 °C. The glass wool was then washed 3 times in assay buffer and twice in distilled H2O and allowed to drain without drying. Trypsination and Size Fractionation The washed siliconized glass wool containing the adsorbed radiolabelled methyltransferase was immersed in 20ml lOmM NH4HCO3 pH 8.1, lmM CaCl2, 0.05% Tween 20 containing 0.1/ig/ml trypsin and incubated at 20°C for 16 hours. The trypzinised sample was concentrated by evaporation under vacuum to a final volume of 1.1 ml and precipitated material was removed by centrifugation. In order to separate intact trypsin from the shorter peptides, the sample was applied to a column (38cm x lcm diam) of Sephadex G25 (superfine) equilibrated with lOmM NH4HCO3 pH 8.1. A symmetrical radioactive peak which eluted at 1.3x the void volume was collected and evaporated to dryness in a vacuum desiccator. This peak contained approximately lOOpmole methylated peptide as estimated from its [3H] content. This step also effectively removed very short peptides. Ion Exchange Chromatography (FPLC) of Peptides The Sephadex G25-purified dried sample was dissolved in 0.5ml buffer D (20mM 2[N-morpholino]ethanesulfonic acidNaOH pH 6.0, 0.02% Tween 20) and subjected to FPLC using a MonoS cation exchanger (5cm x0.5cm diam). The flow rate was 0.5ml/min with a gradient from buffer D to E (buffer D containing 0.4M NaCl) over 60 min. The main radioactive peak Nucleic Acids Research 19 appeared at a NaCl concentration of 0.12M with a minor peak at 0.14M. The fractions corresponding to the main peak of methylated peptide (about 35 pmole) were further purified by HPLC. HPLC The sample was prepared for HPLC by adsorption to a SEPPAK C-18 cartridge (Waters) equilibrated with 50mM NH4AC pH 7.0, washed with 20% acetonitrile and the peptide eluted with 40% acetonitrile in the same buffer. The sample was then lyophilised, dissolved in lO/il 70% formic acid, adjusted to 100/d in Buffer F (1 % acetonitrile, 0.08% trifluoroacetic acid (TFA)) and applied to an Aquapore ODS C-18 column (22cm X0.2 lcm diam., Brownlee Labs) equilibrated in buffer F. The column was eluted at a flow rate of 0.4ml/min with a gradient of 0 - 6 0 % buffer G (90% acetonitrile in 0.06% TFA) over 70min. lml fractions were collected and the two fractions containing radioactive material were retained and separately lyophilised. Amino Acid Sequencing The samples were dissolved in 30/tl 0.1 % TFA, adsorbed to Polyprene-treated glass discs and sequenced using a ABI 477A peptide sequencer. A portion of the sample from each Edman degradation cycle was saved for radioactivity determination. RESULTS Purification of an Oligopeptide Containing the Methyltransferase Active Site In preliminary experiments (data not shown) to purify the methylated form of the methyltransferase, we obtained extremely poor yields of the protein using a variety of experimental approaches. Although the methylated form of die protein does not bind to glass, it exhibits an exaggerated tendency to adhere to hydrophobic surfaces even in the presence of surface active non-ionic detergents. Since this tendency to interact with hydrophobic surfaces— in particular with siliconized glass— was not shared by the contaminating proteins in the partially purified material, we used this property to effect a further purification. A quantitative recovery of the intact methylated protein after its adsorbtion to a siliconized glass support could be achieved only by the use of high concentrations of ionic detergent (1 % SDS), the presence of which severely limited the possibilities for further purification. In contrast, radioactivity from the specifically labelled methylated protein could be recovered in >90% yield following a digestion with trypsin in the presence of the nonionic detergent Tween 20. This observation formed the basis of the purification of a tryptic peptide containing the active cysteine residue of the methyltransferase. Approximately 60pmole of partially (500-fold) purified methyltransferase was incubated with [3H]-substrate DNA containing 70 pmole [3H]m6-Gua and 540pmole enzyme was incubated with an excess of non-radioactive m6-Gua-containing double-stranded oligonucleotide. The reaction mixtures were combined and supplemented with siliconized glass wool to which the methylated enzyme was allowed to adsorb. Non-adsorbed proteins were removed, along with the substrate DNA and oligonucleotide, by extensive washing with assay buffer followed by water. In preliminary experiments, SDS-PAGE and protein determination were used to monitor the composition of the starting material and the material which bound to glass wool and could be removed by 1% SDS. This analysis indicated that >90% of the contaminating proteins were removed in this step. The radioactive protein was removed from the glass wool by trypsin digestion. The glass wool bearing the methylated protein was submerged in a solution containing 0.05% Tween 20 and 0.1/tg/ml trypsin and the mixture incubated at 20°C overnight. The resulting tryptic digest was applied to a column of Sephadex G25 to separate the trypsin from the oligopeptides. Trypsin eluted in the void volume (VJ of the column whereas the radioactive material was included and eluted as a symmetrical peak at approximately 1.3xV 0 . This material was retained and further fractionated by FPLC using a MonoS cation exchange column. Figure 1 shows the elution pattern from the MonoS column. The majority of the radioactive material eluted as a sharp peak at a NaCl concentration of 0.12M with a small shoulder appearing at around 0.14M NaCl. Fractions from die major radioactive peak eluting at 0.12 MNaCl were pooled and further purified by reverse phase HPLC using an Aquapore ODS C-18 column. This procedure resolved the complex mixture of peptides into a number of peaks which absorbed at 220nm (Figure 2). However, all the radioactivity was recovered in two fractions coincident with a single peak of absorbance. The two radioactive fractions which contained respectively 12 and 6pmoles of methylated peptide (representing an overall yield of 3%) were separately subjected to amino acid sequence analysis. Amino Acid Sequence of the Active Site Peptide The amino acid sequence of the purified peptide from the fraction from the HPLC column containing 12pmole is shown below: 1 (?) • 2. Asn • 3. Pro 4. He 5. Pro • 6. He (Phe) 7. Leu (Asp) 8. Thr (He) 9. Pro (Gin) The sequence obtained from the fraction containing 6 pmole of peptide provided confirmation of this sequence. In the later sequencing cycles, corresponding to weaker signals, more than one amino acid was detected indicating the presence of a low level of contaminating oligopeptide. Where two amino acids were detected, both are shown. In each case, however, the first is the more likely as judged by its relative abundance. Methylcysteine yields very poor fluorescence following derivatisation and is not detectable under the standard conditions used for automated sequencing. A portion of the sample from each Edman degradation cycle was therefore retained and its [3H]-radioactivity determined separately. Figure 3 shows that no 1 20 40 60 Fraction No Figure 1. Purification of the Radioactively Labelled Active Site Peptide by FPLC. Separation was carried out on a MonoS cation exchange column. Elution buffers contained: 20mM MES NaOH pH 6.0, 0.02% Tween 20 and a gradient of NaCl (0-0.4M) as shown. Fractions (0.5ml) were collected and aliquots (lO/il) were taken for radioactivity determination. Fractions 26—28 were pooled and used for further purification. 20 Nucleic Acids Research 2 Ada Ogt Asn Asn Dat1 Asn Bovine Asn 3 4 5 Leu Ala Pro | lie | Ser Asp Leu Pro Pro | lie | Pro 6 7 8 9 10 11 12 13 lie lie lie Val Val Phe lie Val Val Pro Pro Pro His His His Arg Arg Art, Val Val Val Me Leu Thr Pro Cys Cys Cys Cys Figure 4. Comparisons Among the Active Sites of O6-Methylguanine-DNA Methyltransferases. Ada: The C-terminal O6-Methylguanine-DNA methyltransferase active site of the E. coli Ada protein. Ogt and Datl: The putative active sites of the E. coli Ogt and the B. subtilis Datl proteins. Bovine: The sequence of the purified calf thymus peptide. The methyl accepting cysteine is arbitrarily assigned position 10. Amino acids exhibiting homology to the bovine sequence are shown boxed. DISCUSSION 20 40 Fraction No Figure 2. Reverse Phase HPLC Purification of the Labelled Peptide. Separation was performed on an Aquapore ODS C-18 column (22cmxO.2 lcm diam.). The column was eluted with a linear gradient (0-60%) of 90% acetonitrile/0.06% TFA in 1% acetonitrile/0.08% TFA. The flow-rate was 0.4ml/min over 70min. The absorbance baseline has been arbitrarily set at the bottom of the Figure. Fractions (1 min) were collected and the radioactivity in aliquots was determined. The total radioactivity present in fractions 31 and 32 is indicated by the bars. All other fractions contained only background radioactivity. The two radioactive fractions were independently subjected to amino acid sequence analysis. • ctivit do) A "E 100 (0 o I ± II 150 50 • 1 I /I / I \ \ 10 15 20 Cycle No Figure 3. Localization of the S-[3H]-methyl Cysteine Residue in the Peptide. Fraction 31 from the ODS C-18 column was subjected to automatic amino acid analysis. The radioactivity in aliquots of the fractions from each Edman degradation cycle from the automatic peptide sequencer was determined by liquid scintillation counting. radioactivity was released before the 10th sequenator cycle. More than 60% of the radioactivity was recovered in the 10th cycle. The remaining <40% was eluted in the 1 lth cycle. These data strongly indicate that the acceptor cysteine residue of the methyltransferase follows the proline residue at position 9 in the above sequence. In Figure 4 we present a comparison of the sequence of the m6-Gua methyltransferase active site of the Ada protein of E. coli along with the proposed active sites for two other related methyltransferases; the constitutive Ogt protein of E. coli and the B. subtilis Datl protein. The data reveal a high degree of conservation in the protein sequence around the active sites of these proteins. The conserved amino acids in the bacterial enzymes; the asparagine at position 2, isoleucine at position 6 and proline at position 9 (the methyl acceptor cysteine is arbitrarily assigned position 10) are also conserved in the bovine enzyme. It is of further interest that the amino acids on the Nterminal side of the acceptor cysteine are of a predominantly hydrophobic nature. In this comparison, we have used the experimentally determined most probable amino acids for positions 5—9 of the bovine enzyme. In the case of the leucine residue at position 7, this assignment is also supported by the observation that the radioactive tryptic peptide bound to a MonoS cation exchange column but not to a MonoQ anion exchange column. This behaviour is not compatible with the presence of a negatively charged amino acid, aspartic acid, at position 7. However, if as seems likely, the His-Arg doublet adjacent to the acceptor cysteine in the other three methyltransferases is conserved in the bovine enzyme, this would introduce two positively charged amino acids into the peptide. A peptide of this sequence would still retain an overall positive charge with Asp assigned to position 7 and would be expected to exhibit the observed behaviour on ion exchange chromatography. We note, however, that the presence of Tween 20 is necessary to prevent irreversible binding of the peptide to both the anion and cation exchangers, most probably as a result of hydrophobic interactions. For this reason, we consider the assignment of Leu to position 7 to be most likely since it is more compatible with the hydrophobic nature of this region of the peptide. While the assignment of isoleucine to position 8 would represent a closer homology to the sequence of the E. coli Ada protein, the overall hydrophobic nature of the amino acid sequence preceding the acceptor cysteine would nevertheless be maintained by the presence of a threonine residue in this position. The substantial homology between the active sites of these proteins probably reflects a conservation of their reaction mechanism. The Datl protein and the Ogt protein exhibit considerable homology (approaching 50%) elsewhere in the primary sequence. Similarly, the carboxy-terminal domain of the Ada protein which contains the m6-Gua methyltransferase active site is highly homologous to both the Ogt and Datl proteins (13,14). In fact, a C-terminal fragment of the Ada protein which Nucleic Acids Research 21 is similar in size to both Ogt and Datl can function efficiently as an m6-Gua methyltransferase in its own right (20). Despite the close resemblances among the bacterial protein sequences and the similarity we have observed at the active site region of the bovine enzyme, it seems likely that such a high degree of conservation between the prokaryotic and mammalian enzymes around the active site of the methyltransferase merely reflects the nature of the methyltransfer reaction, in particular the mobilisation of the methyl group, and is unlikely to extend throughout the protein. In a recent comparison of the known sequences of methyltransferases which catalyse the methylation of cytosine residues in DNA, Posfai et al (21) observed that a ProCys doublet was present in a block of relatively invariant amino acids around the putative active site of the cytosine methyltransferases. It is of interest that, in the cytosine methyltransferases, the amino acid preceding the ProCys doublet by five amino acids is either Leu or He. Furthermore, the ProCys doublet is preceded by a block of hydrophobic amino acids. Both of these structural features are present in the active site region of the m6-Gua-DNA methyltransferases. Thus it seems likely that the particular energetic requirements of methyl group transfer will impose a strong selective pressure for conservation of the active site region of the methyltransferases. Such selective pressure may not apply to the remainder of the protein sequence. In support of this idea is the observation that both monoclonal antibodies and polyclonal antisera raised against the purified C-terminal domain of the E. coli Ada protein, which bears the m6-Gua DNA methyltransferase function, do not cross-react with the human or bovine methyltransferases on immunoblots (J.Hall, B. Sedgwick, unpublished data). An unambiguous assignment of the active cysteine residues of a suicidal methyltransferase has so far only been made for the two domains of the E. coli Ada protein (22,23). While the conserved sequences among the other bacterial enzymes strongly suggest the location of their active sites, direct evidence is lacking. The establishment of the active site sequence of the bovine methyltransferase reported here will, despite its ambiguities, enable an unequivocal assignment of the active site to be made when the full sequence of the protein becomes available. ACKNOWLEDGEMENTS We thank Claire Stephenson for skilled technical assistance, Iain Goldsmith for oligonucleotide synthesisis and Ron Brown for the amino acid sequence analysis. REFERENCES 1. Singer, B. and Grunberger, D. (1983) Molecular Biology of Carcinogens and Mutagens. Plenum Press, New York. 2. Loechler, E., Green, C. L. and Essigmann, J. M. (1984) Proc. Nail Acad. Sci. (USA), 81, 6271-6275. 3. Hill-Perkins, M., Jones, M. D. and Karran, P. (1986) Mutat. Res.,162, 153-163. 4. Abbott, P. J. and Saffhill, R. (1979) Biochim. Biophys Ada, 562, 5 1 - 6 1 . 5. Day, R. S., Ziolkowski, C. H. J., Scudiero, D. A., Meyer, S., Lubiniecki, A. S., Girardi, A. J., Galloway, S. M. and Bynum, G. D. (1980) Nature, 288, 724-727. 6. Sklar, R. and Strauss, B. (1981) Nature, 289, 417-420. 7. Ishizaki, K., Tsujimura, T., Fujio, C , Yangpei, Z., Yawata, H., Nakabeppu, Y., Sekiguchi, M. and Ikenaga, M. (1987) Mutat. Res., 184, 121-128. 8. White, G. R. M., Ockey, C. H., Brennand, J. and Margison, G.P. (\9U)Carcinogenesis, 7, 2077-2080. 9. Hall, J., Kataoka, H., Stephenson, C. and Karran, P. (1988) Carcinogenesis, 9, 1587-1593. 10. Lindahl, T., Sedgwick, B., Sekiguchi, M. and Nakabeppu, Y. (1988) Ann. Rev. Biochem., 57, 133-157. 11. Ather, A., Ahmed, Z. and Riazuddin, S. (1984) Nucleic Acids Res., 12, 2111-2126. 12. Morohoshi, F. and Munakata, N. (1987)7. Bacteriol., 169, 587-592. 13. Potter, P.M., Wilkinson M. C , Fitton, J., Carr, F. J., Brennand, J., Cooper, D. P. and Margison, G. P. (1987) Nucleic Acids Res., 15, 9177-9193. 14. Morohoshi, F., Hayashi, K. and Munakata, N. (1989) Nucleic Acids Res., 17, 6531-6543. 15. Hora, J. F., Eastman, A. and Bresnick, E. (1983) Biochemistry, 22, 3759-3763. 16. Pegg, A. E., Weist, L., Foote, R. S., Mitra, S. and Perry, W. (1983) J. Biol. Chem., 258, 2327-2333. 17. Harris, A. L., Karran, P. and Lindahl, T. (1983) Cancer Res., 43, 3247-3252. 18. Hall, J. and Karran, P. (1986) In: Myrnes, B. and Krokan, H. (eds), Repair of DNA Lesions Introduced by N-Nitroso Compounds. Norwegian University Press, pp. 73-88. 19. Karran, P., Lindahl, T. and Griffin, B. (1979) Nature, 280, 76-77. 20. Demple, B., Jacobsson, A., Olsson, M., Robins, P. and Lindahl, T. (1982) J. Biol. Chem., 257, 13776-13780. 21. Posfai, J., Bhagwat, A. S., Posfai, G. and R. J. Roberts (1989) Nucleic Acids Res., 17, 2421-2435. 22. Demple, B., Sedgwick, B., Robins, P., Totty, N., Waterfield, M. and Lindahl, T. (1985) Proc. Natl. Acad. Sci. (USA), 82, 2688-2692. 23. Sedgwick, B., Robins, P., Totty, N. and Lindahl, T. (1988)7. Biol. Chem., 263, 4430-4433.