Hwang Cheol-Sang, Shemorry Anna, Varshavsky Alexander
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2142-7. doi: 10.1073/pnas.0812316106. Epub 2009 Jan 21.
O(6)-methylguanine (O(6)meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. O(6)meG is repaired through its demethylation by the O(6)-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated O(6) in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system.
O(6)-甲基鸟嘌呤（O(6)meG）以及双链DNA中鸟嘌呤的相关修饰是功能上严重的损伤，可由许多烷基化剂产生，包括强效致癌物N-甲基-N'-硝基-N-亚硝基胍（MNNG）。O(6)meG通过O(6)-烷基鸟嘌呤-DNA烷基转移酶（AGT）的去甲基化作用进行修复。该蛋白在哺乳动物中称为Mgmt（或MGMT），在酿酒酵母中称为Mgt1。AGT蛋白通过将加合物转移到活性位点的半胱氨酸残基上，从鸟嘌呤中烷基化的O(6)上去除甲基和其他烷基。由此产生的AGT的S-烷基-Cys不会恢复为Cys，因此这类修复蛋白只能发挥一次作用。我们在此报告，酿酒酵母Mgt1通过其N端附近的一个降解子，被2种泛素介导的蛋白水解系统共同靶向降解，即Ubr1/Rad6依赖性的N端规则途径和Ufd4/Ubc4依赖性的泛素融合降解（UFD）途径。这些途径对Mgt1的共同靶向作用具有协同性，因为它不仅增加了多聚泛素化Mgt1的产量，还提高了多聚泛素化的持续性。N端规则途径和UFD途径共同介导了Mgt1的组成型和MNNG加速降解。缺乏Ubr1和Ufd4泛素连接酶的酵母细胞对MNNG具有高抗性，但对过表达的Mgt1的毒性敏感。我们考虑了这一发现对DNA修复控制以及泛素系统底物靶向机制的影响。
