Brent T P, Remack J S
Department of Biochemical and Clinical Pharmacology, St Jude Children's Research Hospital, Memphis, TN 38101.
Nucleic Acids Res. 1988 Jul 25;16(14B):6779-88. doi: 10.1093/nar/16.14.6779.
Repair of chloroethylnitrosourea (CENU)-induced precursors of DNA interstrand cross-links by O6-alkylguanine-DNA alkyltransferase (GAT or GATase) appears to be a factor in tumor resistance to therapy with this class of antineoplastic drugs. Since human GAT is highly specific for O6-guanine, yet the probable cross-link structure is N'-Guanine N3-cytosine ethane, rearrangement of the initial O6-guanine adduct via O6,N1ethanoguanine has been proposed. We suggested that GAT reaction with this intermediate would produce DNA covalently linked to protein through an ethane link from N1-guanine to the alkylacceptor site on GAT. In preliminary studies we demonstrated a covalent complex between GAT and carmustine (BCNU)-treated DNA by a precipitation assay method. We have now developed a method for isolating the reaction product of BCNU-treated synthetic 14-mer [32P]-labeled oligodeoxynucleotide and GAT using polyacrylamide gel electrophoresis. This approach can be used to characterize the adducts induced by CENUs that lead to complex formation with GAT. Results obtained to date are consistent with these adducts being precursors of DNA interstrand cross-links.
O6-烷基鸟嘌呤-DNA烷基转移酶（GAT或GATase）对氯乙基亚硝基脲（CENU）诱导的DNA链间交联前体的修复似乎是肿瘤对这类抗肿瘤药物治疗产生抗性的一个因素。由于人GAT对O6-鸟嘌呤具有高度特异性，而可能的交联结构是N'-鸟嘌呤N3-胞嘧啶乙烷，因此有人提出通过O6,N1-乙醇鸟嘌呤对初始O6-鸟嘌呤加合物进行重排。我们认为GAT与该中间体的反应会通过从N1-鸟嘌呤到GAT上烷基受体位点的乙烷连接产生与蛋白质共价连接的DNA。在初步研究中，我们通过沉淀测定法证明了GAT与卡莫司汀（BCNU）处理的DNA之间存在共价复合物。我们现在开发了一种方法，使用聚丙烯酰胺凝胶电泳分离BCNU处理的合成14聚体[32P]标记的寡脱氧核苷酸与GAT的反应产物。这种方法可用于表征由CENU诱导的导致与GAT形成复合物的加合物。迄今为止获得的结果与这些加合物是DNA链间交联前体的观点一致。
