Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.
Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.
复制标题
DOI：
--
复制
发表时间：
1992-11
期刊：
影响因子：
11.2
通讯作者：
Prescilla E. Gonzaga;Philip M. Potter;T. Niu;Dong Yu;D. Ludlum;Joseph A Rafferty;G. P. Margison;T. Brent
中科院分区：
文献类型：
--
作者：
Prescilla E. Gonzaga;Philip M. Potter;T. Niu;Dong Yu;D. Ludlum;Joseph A Rafferty;G. P. Margison;T. Brent
氯乙基亚硝基脲在 DNA 中诱导反应性 O6-鸟嘌呤加合物，该加合物可以形成链间交联或与 DNA 修复蛋白 O6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT) 形成共价复合物。为了检验我们的假设，即这些终产物是由共同前体 1-O6-乙醇鸟嘌呤形成的，我们将链间交联形成的动力学与 MGMT 复合物形成能力的衰减动力学进行了比较。这些过程的半衰期是相同的。我们的假设还预测DNA和MGMT之间的连接是1-(guan-1-yl)-2-(半胱氨酸-S-yl)乙烷。通过与 35S 标记的重组人 MGMT 和氯乙基亚硝基脲处理的寡脱氧核苷酸形成复合物来测试这一概念。通过脱嘌呤和蛋白水解消化降解后，通过使用高效液相色谱和紫外光谱法与合成标记化合物进行比较，确认了[35S]半胱氨酸-鸟嘌呤键的身份。这些结果强化了 DNA 链间交联和 DNA-MGMT 复合物均源自同一前体的假设。数据还表明，1-O6-乙醇鸟嘌呤是 MGMT 的良好底物，因此，在体内某些条件下，DNA-MGMT 复合物的形成可能构成显着的继发性损伤。
Chloroethylnitrosoureas induce reactive O6-guanine adducts in DNA that can form either interstrand cross-links or a covalent complex with the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). To test our hypothesis that these end-products are formed from the common precursor, 1-O6-ethanoguanine, we compared the kinetics of interstrand cross-link formation with those of decay of MGMT complex forming capacity. The half-lives of these processes were identical. Our hypothesis also predicts that the linkage between DNA and MGMT is 1-(guan-1-yl)-2-(cystein-S-yl)ethane. This notion was tested by forming the complex with 35S-labeled recombinant human MGMT and a chloroethylnitrosourea-treated oligodeoxynucleotide. After degradation by depurination and proteolytic digestion, the identity of the [35S]cysteine-guanine linkage was confirmed by comparison with the synthetic marker compound using high performance liquid chromatography and UV spectrometry. These results strengthen the hypothesis that DNA interstrand cross-links and DNA-MGMT complex both arise from the same precursor. The data also suggest that 1-O6-ethanoguanine is a good substrate for MGMT such that, under certain conditions in vivo, DNA-MGMT complex formation may constitute a significant secondary lesion.