Hansen W K, Deutsch W A, Yacoub A, Xu Y, Williams D A, Kelley M R
Department of Pediatrics,Wells Center for Pediatric Research, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808, USA.
J Biol Chem. 1998 Jan 9;273(2):756-62. doi: 10.1074/jbc.273.2.756.
A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow. To overcome the myelosuppression observed by chemotherapeutic alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells. Toward this goal, we have constructed a human fusion protein consisting of O6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O6-methylguanine and apurinic/apyrimidinic (AP) site repair as determined by biochemical analysis. The chimeric protein protected AP endonuclease-deficient Escherichia coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O6-methylguanine DNA methyltransferase-apurinic endonuclease protein resulted in the retention of O6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity. These results demonstrate that the fusion of O6-methylguanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the repair activities of the individual repair proteins. These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinical models of chemotherapy toxicity.
某些化疗烷化剂的剂量限制性毒性在于它们对非靶组织（如骨髓）的毒性作用。为了克服化疗烷化剂所观察到的骨髓抑制，一种方法是提高造血干细胞和祖细胞中DNA修复蛋白的水平。为了实现这一目标，我们构建了一种人融合蛋白，它由O6-甲基鸟嘌呤DNA甲基转移酶与一种脱嘌呤内切酶偶联而成，生化分析表明该融合蛋白对于O6-甲基鸟嘌呤和脱嘌呤/脱嘧啶（AP）位点修复均具有完整功能。这种嵌合蛋白保护缺乏AP内切酶的大肠杆菌细胞免受甲磺酸甲酯和过氧化氢（H2O2）的损伤。表达该嵌合蛋白的逆转录病毒构建体也保护HeLa细胞免受1,3-双（2-氯乙基）-1-亚硝基脲和甲磺酸甲酯的细胞毒性，无论这些试剂是单独使用还是联合使用。此外，正如先前分析所预测的，截短O6-甲基鸟嘌呤DNA甲基转移酶-脱嘌呤内切酶蛋白的脱嘌呤内切酶部分的前150个氨基酸，导致保留了O6-甲基鸟嘌呤DNA甲基转移酶活性，但丧失了所有AP内切酶活性。这些结果表明，将O6-甲基鸟嘌呤DNA甲基转移酶和脱嘌呤内切酶蛋白融合成一种单一的联合修复蛋白可产生一种保留单个修复蛋白修复活性的完整功能蛋白。这些以及其他相关构建体可能对保护敏感组织有用，因此，是在化疗毒性临床前模型中进行测试的候选构建体。
