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. 1987 Oct;6(10):3177–3183. doi: 10.1002/j.1460-2075.1987.tb02629.x

Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein.

S Boiteux 1, T R O'Connor 1, J Laval 1
PMCID: PMC553760  PMID: 3319582

Abstract

An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy. One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain. The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10). After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein. From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS-polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd.

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Selected References

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

  1. Beranek D. T., Weis C. C., Evans F. E., Chetsanga C. J., Kadlubar F. F. Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. Biochem Biophys Res Commun. 1983 Jan 27;110(2):625–631. doi: 10.1016/0006-291x(83)91195-6. [DOI] [PubMed] [Google Scholar]
  2. Boiteux S., Belleney J., Roques B. P., Laval J. Two rotameric forms of open ring 7-methylguanine are present in alkylated polynucleotides. Nucleic Acids Res. 1984 Jul 11;12(13):5429–5439. doi: 10.1093/nar/12.13.5429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boiteux S., Huisman O., Laval J. 3-Methyladenine residues in DNA induce the SOS function sfiA in Escherichia coli. EMBO J. 1984 Nov;3(11):2569–2573. doi: 10.1002/j.1460-2075.1984.tb02175.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boiteux S., Laval J. Imidazole open ring 7-methylguanine: an inhibitor of DNA synthesis. Biochem Biophys Res Commun. 1983 Jan 27;110(2):552–558. doi: 10.1016/0006-291x(83)91185-3. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Breimer L. H. Enzymatic excision from gamma-irradiated polydeoxyribonucleotides of adenine residues whose imidazole rings have been ruptured. Nucleic Acids Res. 1984 Aug 24;12(16):6359–6367. doi: 10.1093/nar/12.16.6359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chetsanga C. J., Frenette G. P. Excision of aflatoxin B1-imidazole ring opened guanine adducts from DNA by formamidopyrimidine-DNA glycosylase. Carcinogenesis. 1983 Aug;4(8):997–1000. doi: 10.1093/carcin/4.8.997. [DOI] [PubMed] [Google Scholar]
  8. Chetsanga C. J., Lindahl T. Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli. Nucleic Acids Res. 1979 Aug 10;6(11):3673–3684. doi: 10.1093/nar/6.11.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chetsanga C. J., Lozon M., Makaroff C., Savage L. Purification and characterization of Escherichia coli formamidopyrimidine-DNA glycosylase that excises damaged 7-methylguanine from deoxyribonucleic acid. Biochemistry. 1981 Sep 1;20(18):5201–5207. doi: 10.1021/bi00521a016. [DOI] [PubMed] [Google Scholar]
  10. Chetsanga C. J., Polidori G., Mainwaring M. Analysis and excision of ring-opened phosphoramide mustard-deoxyguanine adducts in DNA. Cancer Res. 1982 Jul;42(7):2616–2621. [PubMed] [Google Scholar]
  11. 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]
  12. Dürwald H., Hoffmann-Berling H. Endonuclease-I-deficient and ribonuclease I-deficient Escherichia coli mutants. J Mol Biol. 1968 Jul 14;34(2):331–346. doi: 10.1016/0022-2836(68)90257-x. [DOI] [PubMed] [Google Scholar]
  13. Gilson E., Rousset J. P., Clément J. M., Hofnung M. A subfamily of E. coli palindromic units implicated in transcription termination? Ann Inst Pasteur Microbiol. 1986 Nov-Dec;137B(3):259–270. doi: 10.1016/s0769-2609(86)80116-8. [DOI] [PubMed] [Google Scholar]
  14. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hudson G. S., Davidson B. E. Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12. J Mol Biol. 1984 Dec 25;180(4):1023–1051. doi: 10.1016/0022-2836(84)90269-9. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kataoka H., Yamamoto Y., Sekiguchi M. A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate. J Bacteriol. 1983 Mar;153(3):1301–1307. doi: 10.1128/jb.153.3.1301-1307.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lagravère C., Malfoy B., Leng M., Laval J. Ring-opened alkylated guanine is not repaired in Z-DNA. 1984 Aug 30-Sep 5Nature. 310(5980):798–800. doi: 10.1038/310798a0. [DOI] [PubMed] [Google Scholar]
  21. Laval J. Two enzymes are required from strand incision in repair of alkylated DNA. Nature. 1977 Oct 27;269(5631):829–832. doi: 10.1038/269829a0. [DOI] [PubMed] [Google Scholar]
  22. Lawley P. D., Orr D. J. Specific excision of methylation products from DNA of Escherichia coli treated with N-methyl-N'-nitro-N-nitrosoguanidine. Chem Biol Interact. 1970 Aug;2(2):154–157. doi: 10.1016/0009-2797(70)90047-5. [DOI] [PubMed] [Google Scholar]
  23. Lederer F., Ghrir R., Guiard B., Cortial S., Ito A. Two homologous cytochromes b5 in a single cell. Eur J Biochem. 1983 Apr 15;132(1):95–102. doi: 10.1111/j.1432-1033.1983.tb07330.x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Margison G. P., Pegg A. E. Enzymatic release of 7-methylguanine from methylated DNA by rodent liver extracts. Proc Natl Acad Sci U S A. 1981 Feb;78(2):861–865. doi: 10.1073/pnas.78.2.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morel A., Chang J. Y., Cohen P. The complete amino-acid sequence of anglerfish somatostatin-28 II. A new octacosapeptide containing the (Tyr7, Gly10) derivative of somatostatin-14 I. FEBS Lett. 1984 Sep 17;175(1):21–24. doi: 10.1016/0014-5793(84)80561-x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. doi: 10.1073/pnas.72.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sakumi K., Nakabeppu Y., Yamamoto Y., Kawabata S., Iwanaga S., Sekiguchi M. Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli. J Biol Chem. 1986 Nov 25;261(33):15761–15766. [PubMed] [Google Scholar]
  32. 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]
  33. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  34. Steinum A. L., Seeberg E. Nucleotide sequence of the tag gene from Escherichia coli. Nucleic Acids Res. 1986 May 12;14(9):3763–3772. doi: 10.1093/nar/14.9.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Valerie K., Henderson E. E., deRiel J. K. Identification, physical map location and sequence of the denV gene from bacteriophage T4. Nucleic Acids Res. 1984 Nov 12;12(21):8085–8096. doi: 10.1093/nar/12.21.8085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]

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