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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 May;82(9):2688–2692. doi: 10.1073/pnas.82.9.2688

Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis.

B Demple, B Sedgwick, P Robins, N Totty, M D Waterfield, T Lindahl
PMCID: PMC397630  PMID: 3887409

Abstract

The inducible resistance to alkylation mutagenesis and killing in Escherichia coli (the adaptive response) is controlled by the ada gene. The Ada protein acts both as a positive regulator of the response and as a DNA repair enzyme, correcting premutagenic O6-alkylguanine in DNA by suicidal transfer of the alkyl group to one of its own cysteine residues. We have determined the DNA sequence of the cloned ada+ gene and its regulatory region. The data reveal potential sites of ada autoregulation. Amino acid sequence determinations show that the active center for the O6-methylguanine-DNA methyltransferase is located close to the polypeptide COOH terminus and has the unusual sequence -Pro-Cys-His-, preceded by a very hydrophobic region. These same structural features are present at the active site of thymidylate synthase, suggesting a common chemical mechanism for activation of the cysteine.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berman M. L., Landy A. Promoter mutations in the transfer RNA gene tyrT of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4303–4307. doi: 10.1073/pnas.76.9.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Coulondre C., Miller J. H. Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J Mol Biol. 1977 Dec 15;117(3):577–606. doi: 10.1016/0022-2836(77)90059-6. [DOI] [PubMed] [Google Scholar]
  3. Demple B., Jacobsson A., Olsson M., Robins P., Lindahl T. Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase. J Biol Chem. 1982 Nov 25;257(22):13776–13780. [PubMed] [Google Scholar]
  4. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Doolittle R. F. Similar amino acid sequences: chance or common ancestry? Science. 1981 Oct 9;214(4517):149–159. doi: 10.1126/science.7280687. [DOI] [PubMed] [Google Scholar]
  6. Downward J., Yarden Y., Mayes E., Scrace G., Totty N., Stockwell P., Ullrich A., Schlessinger J., Waterfield M. D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature. 1984 Feb 9;307(5951):521–527. doi: 10.1038/307521a0. [DOI] [PubMed] [Google Scholar]
  7. Drenth J., Jansonius J. N., Koekoek R., Swen H. M., Wolthers B. G. Structure of papain. Nature. 1968 Jun 8;218(5145):929–932. doi: 10.1038/218929a0. [DOI] [PubMed] [Google Scholar]
  8. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  9. Evensen G., Seeberg E. Adaptation to alkylation resistance involves the induction of a DNA glycosylase. Nature. 1982 Apr 22;296(5859):773–775. doi: 10.1038/296773a0. [DOI] [PubMed] [Google Scholar]
  10. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  11. Green P. R., Vanaman T. C., Modrich P., Bell R. M. Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase. J Biol Chem. 1983 Sep 25;258(18):10862–10866. [PubMed] [Google Scholar]
  12. Hall J. A., Saffhill R. The incorporation of O6-methyldeoxyguanosine and O4-methyldeoxythymidine monophosphates into DNA by DNA polymerases I and alpha. Nucleic Acids Res. 1983 Jun 25;11(12):4185–4193. doi: 10.1093/nar/11.12.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harris A. L., Karran P., Lindahl T. O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair-deficient cells. Cancer Res. 1983 Jul;43(7):3247–3252. [PubMed] [Google Scholar]
  14. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hochschild A., Irwin N., Ptashne M. Repressor structure and the mechanism of positive control. Cell. 1983 Feb;32(2):319–325. doi: 10.1016/0092-8674(83)90451-8. [DOI] [PubMed] [Google Scholar]
  16. Hora J. F., Eastman A., Bresnick E. O6-methylguanine methyltransferase in rat liver. Biochemistry. 1983 Aug 2;22(16):3759–3763. doi: 10.1021/bi00285a007. [DOI] [PubMed] [Google Scholar]
  17. Jeggo P., Defais T. M., Samson L., Schendel P. An adaptive response of E. coli to low levels of alkylating agent: comparison with previously characterised DNA repair pathways. Mol Gen Genet. 1977 Nov 29;157(1):1–9. doi: 10.1007/BF00268680. [DOI] [PubMed] [Google Scholar]
  18. Jeggo P. Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents. J Bacteriol. 1979 Sep;139(3):783–791. doi: 10.1128/jb.139.3.783-791.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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 Apr 22;296(5859):770–773. doi: 10.1038/296770a0. [DOI] [PubMed] [Google Scholar]
  20. Kataoka H., Sekiguchi M. Molecular cloning and characterization of the alkB gene of Escherichia coli. Mol Gen Genet. 1985;198(2):263–269. doi: 10.1007/BF00383004. [DOI] [PubMed] [Google Scholar]
  21. Lindahl T., Demple B., Robins P. Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase. EMBO J. 1982;1(11):1359–1363. doi: 10.1002/j.1460-2075.1982.tb01323.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loechler E. L., Green C. L., Essigmann J. M. In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6271–6275. doi: 10.1073/pnas.81.20.6271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maley F., Belfort M., Maley G. Probing the infra-structure of thymidylate synthase and deoxycytidylate deaminase. Adv Enzyme Regul. 1984;22:413–430. doi: 10.1016/0065-2571(84)90023-2. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. May M. S., Hattman S. Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes. J Bacteriol. 1975 Aug;123(2):768–770. doi: 10.1128/jb.123.2.768-770.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McCarthy J. G., Edington B. V., Schendel P. F. Inducible repair of phosphotriesters in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7380–7384. doi: 10.1073/pnas.80.24.7380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCarthy T. V., Karran P., Lindahl T. Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli. EMBO J. 1984 Mar;3(3):545–550. doi: 10.1002/j.1460-2075.1984.tb01844.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakabeppu Y., Miyata T., Kondo H., Iwanaga S., Sekiguchi M. Structure and expression of the alkA gene of Escherichia coli involved in adaptive response to alkylating agents. J Biol Chem. 1984 Nov 25;259(22):13730–13736. [PubMed] [Google Scholar]
  29. Olsson M., Lindahl T. Repair of alkylated DNA in Escherichia coli. Methyl group transfer from O6-methylguanine to a protein cysteine residue. J Biol Chem. 1980 Nov 25;255(22):10569–10571. [PubMed] [Google Scholar]
  30. Robins P., Cairns J. Quantitation of the adaptive response to alkylating agents. Nature. 1979 Jul 5;280(5717):74–76. doi: 10.1038/280074a0. [DOI] [PubMed] [Google Scholar]
  31. Robins P., Harris A. L., Goldsmith I., Lindahl T. Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase. Nucleic Acids Res. 1983 Nov 25;11(22):7743–7758. doi: 10.1093/nar/11.22.7743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Samson L., Cairns J. A new pathway for DNA repair in Escherichia coli. Nature. 1977 May 19;267(5608):281–283. doi: 10.1038/267281a0. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sedgwick B. Genetic mapping of ada and adc mutations affecting the adaptive response of Escherichia coli to alkylating agents. J Bacteriol. 1982 May;150(2):984–988. doi: 10.1128/jb.150.2.984-988.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sedgwick B. Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli. Mol Gen Genet. 1983;191(3):466–472. doi: 10.1007/BF00425764. [DOI] [PubMed] [Google Scholar]
  36. Sukumar S., Notario V., Martin-Zanca D., Barbacid M. Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature. 1983 Dec 15;306(5944):658–661. doi: 10.1038/306658a0. [DOI] [PubMed] [Google Scholar]
  37. Teo I., Sedgwick B., Demple B., Li B., Lindahl T. Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage. EMBO J. 1984 Sep;3(9):2151–2157. doi: 10.1002/j.1460-2075.1984.tb02105.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Volkert M. R., Nguyen D. C. Induction of specific Escherichia coli genes by sublethal treatments with alkylating agents. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4110–4114. doi: 10.1073/pnas.81.13.4110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yoo O. J., Powell C. T., Agarwal K. L. Molecular cloning and nucleotide sequence of full-length of cDNA coding for porcine gastrin. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1049–1053. doi: 10.1073/pnas.79.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]

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