Affinity purification and characterization of humanO6-alkylguanine-DNA aikyltransferase complexed with BCNU-treated, synthetic oligonudeotide
- 1 January 1989
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 17 (16) , 6581-6590
- https://doi.org/10.1093/nar/17.16.6581
Abstract
Tumor cells resistant to chloroethylnitrosourea (CENU) therapy contain high levels of O6-alkylguanine DNA-alkytransferase (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-trated, 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 sliver-stained band after SDS-polyacrylamide gel electrophoresis. Autoradiography and Western immunoblotting confirmed that this band contained both the radiolabeled oligonucleotide and the GATase protein.This publication has 22 references indexed in Scilit:
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