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. 1989 Aug 25;17(16):6581–6590. doi: 10.1093/nar/17.16.6581

Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide.

P E Gonzaga 1, T P Brent 1
PMCID: PMC318351  PMID: 2780288

Abstract

Tumor cells resistant to chloroethylnitrosourea (CENU) therapy contain high levels of O6-alkylguanine DNA-alkyltransferase (GATase), a DNA repair enzyme that aborts DNA interstrand cross-linking by removing CENU-induced O6-alkylguanine adducts. Because the transferase binds covalently to CENU-treated oligonucleotides, we reacted partially purified GATase from cultured human lymphoblasts with a BCNU-treated, 35S-5'-end-labeled, synthetic oligonucleotide designed to have a polyadenylated 3' terminus. Immunoprobing Western blots of this reaction mixture with GATase-specific monoclonal antibody indicated that 25-30% of the transferase became complexed. We purified this complex by affinity chromatography with oligo(dT) cellulose, recovering homogenous material that appeared as a discrete 35-kDa Coomassie blue or silver-stained band after SDS-polyacrylamide gel electrophoresis. Autoradiography and Western immunoblotting confirmed that this band contained both the radiolabeled oligonucleotide and the GATase protein.

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Selected References

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

  1. Bogden J. M., Eastman A., Bresnick E. A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA. Nucleic Acids Res. 1981 Jul 10;9(13):3089–3103. doi: 10.1093/nar/9.13.3089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brent T. P., Houghton P. J., Houghton J. A. O6-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea. Proc Natl Acad Sci U S A. 1985 May;82(9):2985–2989. doi: 10.1073/pnas.82.9.2985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brent T. P. Isolation and purification of O6-alkylguanine-DNA alkyltransferase from human leukemic cells. Prevention of chloroethylnitrosourea-induced cross-links by purified enzyme. Pharmacol Ther. 1985;31(1-2):121–140. doi: 10.1016/0163-7258(85)90040-3. [DOI] [PubMed] [Google Scholar]
  4. Brent T. P., Remack J. S. Formation of covalent complexes between human O6-alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligodeoxynucleotides. Nucleic Acids Res. 1988 Jul 25;16(14B):6779–6788. doi: 10.1093/nar/16.14.6779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brent T. P., Smith D. G., Remack J. S. Evidence that O6-alkylguanine-DNA alkyltransferase becomes covalently bound to DNA containing 1,3-bis(2-chloroethyl)-1-nitrosourea-induced precursors of interstrand cross-links. Biochem Biophys Res Commun. 1987 Jan 30;142(2):341–347. doi: 10.1016/0006-291x(87)90279-8. [DOI] [PubMed] [Google Scholar]
  6. Brent T. P. Suppression of cross-link formation in chloroethylnitrosourea-treated DNA by an activity in extracts of human leukemic lymphoblasts. Cancer Res. 1984 May;44(5):1887–1892. [PubMed] [Google Scholar]
  7. Briscoe W. T., Duarte S. P. Preferential alkylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of guanines with guanines as neighboring bases in DNA. Biochem Pharmacol. 1988 Mar 15;37(6):1061–1066. doi: 10.1016/0006-2952(88)90511-4. [DOI] [PubMed] [Google Scholar]
  8. Connors T. A. Alkylating agents, nitrosoureas and alkyltriazenes. Cancer Chemother Biol Response Modif. 1987;9:23–35. [PubMed] [Google Scholar]
  9. Hartley J. A., Gibson N. W., Kohn K. W., Mattes W. B. DNA sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents. Cancer Res. 1986 Apr;46(4 Pt 2):1943–1947. [PubMed] [Google Scholar]
  10. 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]
  11. Ludlum D. B., Mehta J. R., Tong W. P. Prevention of 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane cross-link formation in DNA by rat liver O6-alkylguanine-DNA alkyltransferase. Cancer Res. 1986 Jul;46(7):3353–3357. [PubMed] [Google Scholar]
  12. Mattes W. B., Hartley J. A., Kohn K. W. DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res. 1986 Apr 11;14(7):2971–2987. doi: 10.1093/nar/14.7.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mehta J. R., Ludlum D. B., Renard A., Verly W. G. Repair of O6-ethylguanine in DNA by a chromatin fraction from rat liver: transfer of the ethyl group to an acceptor protein. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6766–6770. doi: 10.1073/pnas.78.11.6766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pegg A. E., Scicchitano D., Morimoto K., Dolan M. E. Specificity of O6-alkylguanine-DNA alkyltransferase. IARC Sci Publ. 1987;(84):30–34. [PubMed] [Google Scholar]
  15. Pegg A. E., Wiest L., Foote R. S., Mitra S., Perry W. Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem. 1983 Feb 25;258(4):2327–2333. [PubMed] [Google Scholar]
  16. Renard A., Verly W. G. A chromatin factor in rat liver which destroys O6-ethylguanine in DNA. FEBS Lett. 1980 May 19;114(1):98–102. doi: 10.1016/0014-5793(80)80868-4. [DOI] [PubMed] [Google Scholar]
  17. Scicchitano D., Jones R. A., Kuzmich S., Gaffney B., Lasko D. D., Essigmann J. M., Pegg A. E. Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase. Carcinogenesis. 1986 Aug;7(8):1383–1386. doi: 10.1093/carcin/7.8.1383. [DOI] [PubMed] [Google Scholar]
  18. Sklar R., Strauss B. Removal of O6-methylguanine from DNA of normal and xeroderma pigmentosum-derived lymphoblastoid lines. Nature. 1981 Jan 29;289(5796):417–420. doi: 10.1038/289417a0. [DOI] [PubMed] [Google Scholar]
  19. Swenberg J. A., Bedell M. A., Billings K. C., Umbenhauer D. R., Pegg A. E. Cell-specific differences in O6-alkylguanine DNA repair activity during continuous exposure to carcinogen. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5499–5502. doi: 10.1073/pnas.79.18.5499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tong W. P., Kirk M. C., Ludlum D. B. Mechanism of action of the nitrosoureas--V. Formation of O6-(2-fluoroethyl)guanine and its probable role in the crosslinking of deoxyribonucleic acid. Biochem Pharmacol. 1983 Jul 1;32(13):2011–2015. doi: 10.1016/0006-2952(83)90420-3. [DOI] [PubMed] [Google Scholar]
  21. Trask D. K., DiDonato J. A., Muller M. T. Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II. EMBO J. 1984 Mar;3(3):671–676. doi: 10.1002/j.1460-2075.1984.tb01865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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