Abstract
DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.
Full Text
The Full Text of this article is available as a PDF (106.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Chan C. L., Wu Z., Ciardelli T., Eastman A., Bresnick E. Kinetic and DNA-binding properties of recombinant human O6-methylguanine-DNA methyltransferase. Arch Biochem Biophys. 1993 Jan;300(1):193–200. doi: 10.1006/abbi.1993.1027. [DOI] [PubMed] [Google Scholar]
- Graves R. J., Li B. F., Swann P. F. Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase. Carcinogenesis. 1989 Apr;10(4):661–666. doi: 10.1093/carcin/10.4.661. [DOI] [PubMed] [Google Scholar]
- 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]
- Miller P. S., Cushman C. D. Selective modification of cytosines in oligodeoxyribonucleotides. Bioconjug Chem. 1992 Jan-Feb;3(1):74–79. doi: 10.1021/bc00013a012. [DOI] [PubMed] [Google Scholar]
- Moore M. H., Gulbis J. M., Dodson E. J., Demple B., Moody P. C. Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli. EMBO J. 1994 Apr 1;13(7):1495–1501. doi: 10.1002/j.1460-2075.1994.tb06410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Pegg A. E., Boosalis M., Samson L., Moschel R. C., Byers T. L., Swenn K., Dolan M. E. Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry. 1993 Nov 16;32(45):11998–12006. doi: 10.1021/bi00096a009. [DOI] [PubMed] [Google Scholar]
- Sassanfar M., Dosanjh M. K., Essigmann J. M., Samson L. Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. Suggestive evidence for O4-methylthymine repair by eukaryotic methyltransferases. J Biol Chem. 1991 Feb 15;266(5):2767–2771. [PubMed] [Google Scholar]
- Sproat B. S., Iribarren A. M., Garcia R. G., Beijer B. New synthetic routes to synthons suitable for 2'-O-allyloligoribonucleotide assembly. Nucleic Acids Res. 1991 Feb 25;19(4):733–738. doi: 10.1093/nar/19.4.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zuckermann R., Corey D., Schultz P. Efficient methods for attachment of thiol specific probes to the 3'-ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Jul 10;15(13):5305–5321. doi: 10.1093/nar/15.13.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]