Dolan M E, Morimoto K, Pegg A E
Cancer Res. 1985 Dec;45(12 Pt 1):6413-7.
Exposure of HeLa cells to 0.2 mM O6-methylguanine for 4 h or longer led to a 70-80% loss in the activity of the DNA-repair protein, O6-alkylguanine-DNA alkyltransferase. The decline in alkyltransferase activity brought about by O6-methylguanine was reversible on removing the base but at least 48 h were required for complete restoration. This loss of activity could also be brought about by other O6-alkylguanines including ethyl, n-propyl, and n-butyl, but the isopropyl and 2-hydroxyethyl derivatives were considerably less active. The rate of decline of alkyltransferase activity produced by O6-methylguanine was much more rapid than the rate of loss when protein synthesis was inhibited indicating that it was not brought about by blocking the synthesis of the protein. The loss of alkyltransferase activity was not prevented by the addition of inhibitors of nucleic acid or protein synthesis suggesting that it did not require protein synthesis or the incorporation of the O6-alkylguanine into nucleic acids. When cell free O6-alkylguanine-DNA alkyltransferase preparations were incubated in vitro with O6-methylguanine they became inactivated and when O6-[3H]methylguanine was used, [3H]guanine was produced. The inactivation was concentration dependent requiring 0.4 mM for a maximal rate and was quite slow requiring 3-4 hours for completion. These results suggest that the loss of O6-alkylguanine-DNA alkyltransferase activity is brought about by the free base acting as a very weak substrate for the protein. Exposure of mammalian cells to O6-methylguanine or O6-n-butylguanine provides a means by which the level of O6-alkylguanine-DNA-alkyltransferase can be regulated experimentally. This should enable the design of experiments to examine the role of O6-alkylguanine adducts in mutagenesis, carcinogenesis, and cell toxicity after administration of alkylating agents.
将HeLa细胞暴露于0.2 mM O6-甲基鸟嘌呤中4小时或更长时间，会导致DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶的活性丧失70-80%。由O6-甲基鸟嘌呤引起的烷基转移酶活性下降在去除该碱基后是可逆的，但完全恢复至少需要48小时。其他O6-烷基鸟嘌呤，包括乙基、正丙基和正丁基，也能导致这种活性丧失，但异丙基和2-羟乙基衍生物的活性要低得多。O6-甲基鸟嘌呤导致的烷基转移酶活性下降速度比蛋白质合成受抑制时的丧失速度快得多，这表明它不是由蛋白质合成受阻引起的。添加核酸或蛋白质合成抑制剂并不能阻止烷基转移酶活性的丧失，这表明它不需要蛋白质合成或O6-烷基鸟嘌呤掺入核酸。当无细胞的O6-烷基鸟嘌呤-DNA烷基转移酶制剂在体外与O6-甲基鸟嘌呤一起孵育时，它们会失活，当使用O6-[3H]甲基鸟嘌呤时，会产生[3H]鸟嘌呤。失活是浓度依赖性的，最大速率需要0.4 mM，而且相当缓慢，需要3-4小时才能完成。这些结果表明，O6-烷基鸟嘌呤-DNA烷基转移酶活性的丧失是由游离碱基作为该蛋白质的一种非常弱的底物引起的。将哺乳动物细胞暴露于O6-甲基鸟嘌呤或O6-正丁基鸟嘌呤提供了一种通过实验调节O6-烷基鸟嘌呤-DNA-烷基转移酶水平的方法。这应该能够设计实验来研究烷基化剂给药后O6-烷基鸟嘌呤加合物在诱变、致癌和细胞毒性中的作用。
