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Nucleic Acids Research, 1989, Vol. 17, No. 16 6581-6590
© 1989

ENZYMOLOGY

Affinity purification and characterization of human O6-alkylguanine-DNA aikyltransferase complexed with BCNU-treated, synthetic oligonudeotide

Prescilla E. Gonzaga and Thomas P. Brent*

Department of Biochemical and Clinical Pharmacology, St Jude Children's Research Hospital 332 North Lauderdale, Memphis, TN 38101, USA

*To whom correspondence should be addressed

Received May 9, 1989. Revised July 13, 1989. Accepted July 13, 1989.

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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