Skip to main content
NCBI home page
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
. 1986 Sep;83(17):6297–6301. doi: 10.1073/pnas.83.17.6297

Regulatory mechanisms for induction of synthesis of repair enzymes in response to alkylating agents: ada protein acts as a transcriptional regulator.

Y Nakabeppu, M Sekiguchi
PMCID: PMC386490  PMID: 3529081

Abstract

Expression of the ada and alkA genes, both of which are involved in the adaptive response of Escherichia coli to alkylating agents, is positively controlled by Ada protein, the product of the ada gene. Large amounts of ada- and alkA-specific RNA were formed in cells treated with a methylating agent, whereas little such RNA was produced in untreated cells. The in vivo transcription-initiation sites for the two genes were determined by primer-extension cDNA synthesis. In an in vitro reconstituted system, both ada and alkA transcripts were formed in an Ada protein-dependent manner. However, responses of the two transcription reactions to methylating agents differed; ada transcription was stimulated by methylnitrosourea, while alkA transcription was suppressed. We prepared a methylated form of Ada protein by an in vitro reaction and compared the activity with that of the normal, unmethylated form. The methylated form was more effective in promoting ada transcription than was the unmethylated form, but the effects of both forms were much the same with regard to alkA transcription. Based on these findings, we propose a model for the molecular mechanism of adaptive response.

Full text

PDF
6297

Images in this article

6298Image
on p.6298
6298Image
on p.6298
6300Image
on p.6300
6301Image
on p.6301
6301Image
on p.6301

Selected References

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

  1. Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes W. M., Bevan M., Son P. H. Kilo-sequencing: creation of an ordered nest of asymmetric deletions across a large target sequence carried on phage M13. Methods Enzymol. 1983;101:98–122. doi: 10.1016/0076-6879(83)01008-3. [DOI] [PubMed] [Google Scholar]
  3. Cathcart R., Goldthwait D. A. Enzymatic excision of 3-methyladenine and 7-methylguanine by a rat liver nuclear fraction. Biochemistry. 1981 Jan 20;20(2):273–280. doi: 10.1021/bi00505a007. [DOI] [PubMed] [Google Scholar]
  4. Clarke N. D., Kvaal M., Seeberg E. Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II. Mol Gen Genet. 1984;197(3):368–372. doi: 10.1007/BF00329931. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Demple B., Sedgwick B., Robins P., Totty N., Waterfield M. D., Lindahl T. Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis. Proc Natl Acad Sci U S A. 1985 May;82(9):2688–2692. doi: 10.1073/pnas.82.9.2688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ebina Y., Nakazawa A. Cyclic AMP-dependent initiation and rho-dependent termination of colicin E1 gene transcription. J Biol Chem. 1983 Jun 10;258(11):7072–7078. [PubMed] [Google Scholar]
  8. 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]
  9. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Kajitani M., Ishihama A. Determination of the promoter strength in the mixed transcription system: promoters of lactose, tryptophan and ribosomal protein L10 operons from Escherichia coli. Nucleic Acids Res. 1983 Feb 11;11(3):671–686. doi: 10.1093/nar/11.3.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lemotte P. K., Walker G. C. Induction and autoregulation of ada, a positively acting element regulating the response of Escherichia coli K-12 to methylating agents. J Bacteriol. 1985 Mar;161(3):888–895. doi: 10.1128/jb.161.3.888-895.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McCarthy T. V., Lindahl T. Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli. Nucleic Acids Res. 1985 Apr 25;13(8):2683–2698. doi: 10.1093/nar/13.8.2683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nakabeppu Y., Kondo H., Kawabata S., Iwanaga S., Sekiguchi M. Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase. J Biol Chem. 1985 Jun 25;260(12):7281–7288. [PubMed] [Google Scholar]
  18. 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 Nov 25;259(22):13723–13729. [PubMed] [Google Scholar]
  19. Nakabeppu Y., Mine Y., Sekiguchi M. Regulation of expression of the cloned ada gene in Escherichia coli. Mutat Res. 1985 Sep;146(2):155–167. doi: 10.1016/0167-8817(85)90006-9. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Otsuka M., Nakabeppu Y., Sekiguchi M. Ability of various alkylating agents to induce adaptive and SOS responses: a study with lacZ fusion. Mutat Res. 1985 Sep;146(2):149–154. doi: 10.1016/0167-8817(85)90005-7. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  25. Schendel P. F., Robins P. E. Repair of O6-methylguanine in adapted Escherichia coli. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6017–6020. doi: 10.1073/pnas.75.12.6017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Treisman R., Proudfoot N. J., Shander M., Maniatis T. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing. Cell. 1982 Jul;29(3):903–911. doi: 10.1016/0092-8674(82)90452-4. [DOI] [PubMed] [Google Scholar]
  29. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  30. Yamamoto Y., Katsuki M., Sekiguchi M., Otsuji N. Escherichia coli gene that controls sensitivity to alkylating agents. J Bacteriol. 1978 Jul;135(1):144–152. doi: 10.1128/jb.135.1.144-152.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES