Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide.
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.
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
P E
PE
Gonzaga
T P
TP
Brent
D001483:Base Sequence; D015153:Blotting, Western; D002330:Carmustine / Q000494:pharmacology*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding
D009838:Oligodeoxyribonucleotides; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine