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. 1992 Aug 1;285(Pt 3):707–709. doi: 10.1042/bj2850707

C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity.

R H Elder 1, J Tumelty 1, K T Douglas 1, G P Margison 1, J A Rafferty 1
PMCID: PMC1132851  PMID: 1497608

Abstract

A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; methylated-DNA: protein-cysteine methyltransferase) has been manipulated to generate a C-terminally deleted protein which retains full methyl-transfer activity. The elimination of 22 amino-acid residues from the C-terminus was achieved by endonuclease-SacI digestion of the 623 bp cDNA coding sequence and ligation of a SacI/HindIII linker containing an in-frame stop codon. The truncated protein was characterized by its reduced molecular mass in immunoblots probed with an antiserum against the full-length protein and by fluorography after incubation with [3H]methylated calf thymus DNA. The rate of methyl transfer was virtually identical for the full-length and truncated ATases. The construction of such a truncated, yet still functional, ATase, with a molecular mass of 19.7 kDa should facilitate a detailed n.m.r. structural study and help to determine the functional significance of the C-terminal domain of mammalian ATases.

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Selected References

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

  1. Fan C. Y., Potter P. M., Rafferty J., Watson A. J., Cawkwell L., Searle P. F., O'Connor P. J., Margison G. P. Expression of a human O6-alkylguanine-DNA-alkyltransferase cDNA in human cells and transgenic mice. Nucleic Acids Res. 1990 Oct 11;18(19):5723–5727. doi: 10.1093/nar/18.19.5723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Hakura A., Morimoto K., Sofuni T., Nohmi T. Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase. J Bacteriol. 1991 Jun;173(12):3663–3672. doi: 10.1128/jb.173.12.3663-3672.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hayakawa H., Koike G., Sekiguchi M. Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA. J Mol Biol. 1990 Jun 20;213(4):739–747. doi: 10.1016/S0022-2836(05)80260-8. [DOI] [PubMed] [Google Scholar]
  4. Margison G. P., Cooper D. P., Brennand J. Cloning of the E. coli O6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay. Nucleic Acids Res. 1985 Mar 25;13(6):1939–1952. doi: 10.1093/nar/13.6.1939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Morten J. E., Margison G. P. Increased O6-alkylguanine alkyltransferase activity in Chinese hamster V79 cells following selection with chloroethylating agents. Carcinogenesis. 1988 Jan;9(1):45–49. doi: 10.1093/carcin/9.1.45. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Pegg A. E., Wiest L., Mummert C., Dolan M. E. Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts. Carcinogenesis. 1991 Sep;12(9):1671–1677. doi: 10.1093/carcin/12.9.1671. [DOI] [PubMed] [Google Scholar]
  8. Potter P. M., Rafferty J. A., Cawkwell L., Wilkinson M. C., Cooper D. P., O'Connor P. J., Margison G. P. Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver. Carcinogenesis. 1991 Apr;12(4):727–733. doi: 10.1093/carcin/12.4.727. [DOI] [PubMed] [Google Scholar]
  9. Potter P. M., Wilkinson M. C., Fitton J., Carr F. J., Brennand J., Cooper D. P., Margison G. P. Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli. Nucleic Acids Res. 1987 Nov 25;15(22):9177–9193. doi: 10.1093/nar/15.22.9177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Rafferty J. A., Elder R. H., Watson A. J., Cawkwell L., Potter P. M., Margison G. P. Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA. Nucleic Acids Res. 1992 Apr 25;20(8):1891–1895. doi: 10.1093/nar/20.8.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Rahden-Staron I., Laval F. cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase. Biochem Biophys Res Commun. 1991 Jun 14;177(2):597–602. doi: 10.1016/0006-291x(91)91830-6. [DOI] [PubMed] [Google Scholar]
  12. Rydberg B., Spurr N., Karran P. cDNA cloning and chromosomal assignment of the human O6-methylguanine-DNA methyltransferase. cDNA expression in Escherichia coli and gene expression in human cells. J Biol Chem. 1990 Jun 5;265(16):9563–9569. [PubMed] [Google Scholar]
  13. Sakumi K., Shiraishi A., Hayakawa H., Sekiguchi M. Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase. Nucleic Acids Res. 1991 Oct 25;19(20):5597–5601. doi: 10.1093/nar/19.20.5597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Santibanez-Koref M., Elder R. H., Fan C. Y., Cawkwell L., McKie J. H., Douglas K. T., Margison G. P., Rafferty J. A. Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties. Mol Carcinog. 1992;5(2):161–169. doi: 10.1002/mc.2940050212. [DOI] [PubMed] [Google Scholar]
  15. Shiota S., von Wronski M. A., Tano K., Bigner D. D., Brent T. P., Mitra S. Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli. Biochemistry. 1992 Feb 25;31(7):1897–1903. doi: 10.1021/bi00122a001. [DOI] [PubMed] [Google Scholar]
  16. Shiraishi A., Sakumi K., Nakatsu Y., Hayakawa H., Sekiguchi M. Isolation and characterization of cDNA and genomic sequences for mouse O6-methylguanine-DNA methyltransferase. Carcinogenesis. 1992 Feb;13(2):289–296. doi: 10.1093/carcin/13.2.289. [DOI] [PubMed] [Google Scholar]
  17. Tano K., Shiota S., Collier J., Foote R. S., Mitra S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. Proc Natl Acad Sci U S A. 1990 Jan;87(2):686–690. doi: 10.1073/pnas.87.2.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Xiao W., Derfler B., Chen J., Samson L. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene. EMBO J. 1991 Aug;10(8):2179–2186. doi: 10.1002/j.1460-2075.1991.tb07753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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