Gonzaga P E, Brent T P
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Nucleic Acids Res. 1989 Aug 25;17(16):6581-90. doi: 10.1093/nar/17.16.6581.
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.
对氯乙基亚硝基脲（CENU）疗法耐药的肿瘤细胞含有高水平的O6-烷基鸟嘌呤DNA烷基转移酶（GATase），这是一种DNA修复酶，可通过去除CENU诱导的O6-烷基鸟嘌呤加合物来阻止DNA链间交联。由于该转移酶与CENU处理的寡核苷酸共价结合，我们使来自培养的人淋巴母细胞的部分纯化的GATase与经BCNU处理的、35S-5'-末端标记的、设计为具有聚腺苷酸化3'末端的合成寡核苷酸反应。用GATase特异性单克隆抗体对该反应混合物进行免疫印迹探测表明，25%-30%的转移酶形成了复合物。我们通过用寡聚（dT）纤维素进行亲和层析纯化该复合物，回收了在SDS-聚丙烯酰胺凝胶电泳后呈现为离散的35 kDa考马斯亮蓝或银染条带的均一物质。放射自显影和蛋白质免疫印迹证实该条带同时包含放射性标记的寡核苷酸和GATase蛋白。
