Liu H, Xu-Welliver M, Pegg A E
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, 17033-0850, USA.
Mutat Res. 2000 Jul 20;452(1):1-10. doi: 10.1016/s0027-5107(00)00062-2.
The expression of the DNA repair protein human O(6)-alkylguanine-DNA alkyltransferase (AGT) in Escherichia coli strains GWR109 or TRG8 that lack endogenous AGT greatly increased the toxicity and mutagenicity of 1,2-dibromoethane (DBE). Pretreatment of strain TRG8 expressing human AGT, which is permeable to exogenous drugs, with the AGT inhibitor O(6)-benzylguanine (BG) abolished the lethal and mutagenic effects of DBE, indicating that an active AGT is required for promoting DBE genotoxicity. This was confirmed by the observation that E. coli expressing either the C145A AGT mutant, which is inactive due to loss of the alkyl acceptor site, or mutants Y114E and R128A, which are inactive due to alteration of the DNA binding domain, did not enhance the action of DBE. However, the AGT mutant protein P138M/V139L/P140K, which is active in repairing methylated DNA but is totally resistant to inactivation by BG due to alterations in the active site pocket, was unable to enhance the genotoxicity of DBE. Similarly, other mutants, G156P, Y158H and K165R that are strongly resistant to BG, were much less effective than wild type AGT in mediating the genotoxicity of DBE. Mutant P140A, which is moderately resistant to BG, did increase mutations in response to DBE but was less active than wild type. These results suggest that human AGT is able to interact with a DNA lesion produced by DBE but, instead of repairing it, converts it to a more genotoxic adduct. This interaction is prevented by mutations that modify the active site of AGT to exclude BG.
缺乏内源性O(6)-烷基鸟嘌呤-DNA烷基转移酶（AGT）的大肠杆菌菌株GWR109或TRG8中，DNA修复蛋白人O(6)-烷基鸟嘌呤-DNA烷基转移酶（AGT）的表达极大地增加了1,2-二溴乙烷（DBE）的毒性和致突变性。用AGT抑制剂O(6)-苄基鸟嘌呤（BG）预处理可渗透外源药物的表达人AGT的TRG8菌株，消除了DBE的致死和致突变作用，表明促进DBE遗传毒性需要活性AGT。这一点通过以下观察得到证实：表达因烷基受体位点缺失而无活性的C145A AGT突变体或因DNA结合域改变而无活性的Y114E和R128A突变体的大肠杆菌，不会增强DBE的作用。然而，在修复甲基化DNA方面有活性但由于活性位点口袋改变而对BG灭活完全抗性的AGT突变蛋白P138M/V139L/P140K，无法增强DBE的遗传毒性。同样，对BG有强抗性的其他突变体G156P、Y158H和K165R，在介导DBE的遗传毒性方面比野生型AGT的效果要差得多。对BG有中度抗性的突变体P140A，在接触DBE时确实会增加突变，但活性低于野生型。这些结果表明，人AGT能够与DBE产生的DNA损伤相互作用，但不是修复它，而是将其转化为更具遗传毒性的加合物。修饰AGT活性位点以排除BG的突变可阻止这种相互作用。
