AFFINITY PURIFICATION AND CHARACTERIZATION OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE COMPLEXED WITH BCNU-TREATED, SYNTHETIC OLIGONUCLEOTIDE
AFFINITY PURIFICATION AND CHARACTERIZATION OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE COMPLEXED WITH BCNU-TREATED, SYNTHETIC OLIGONUCLEOTIDE
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DOI：
10.1093/nar/17.16.6581
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发表时间：
1989-08-25
影响因子：
14.9
通讯作者：
BRENT, TP
中科院分区：
文献类型：
--
作者：
GONZAGA, PE;BRENT, TP
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.
