Function of domains of human O6-alkylguanine-DNA alkyltransferase
Function of domains of human O6-alkylguanine-DNA alkyltransferase
复制标题
DOI：
10.1021/bi051460d
复制
发表时间：
2005-11-22
期刊：
影响因子：
2.9
通讯作者：
Pegg, AE
中科院分区：
文献类型：
--
作者：
Fang, QM;Kanugula, S;Pegg, AE
O-6-烷基鸟嘌呤-DNA烷基转移酶（O-6-Alkylguanine-DNA alkyltransferase，AGT）是一种重要的DNA修复蛋白，通过将O-6-烷基鸟嘌呤转化为鸟嘌呤形成AGT蛋白中的S-甲基半胱氨酸来保护DNA免受烷化剂的侵害。人AGT的晶体结构清楚地表明存在两个结构域。N-末端结构域含有结合的锌原子，并且锌结合赋予修复活性的机械增强，但该结构域没有已知的功能。C-末端结构域包含迄今为止涉及烷基转移的所有残基，包括半胱氨酸受体位点（Cys 145）、O-6-烷基鸟嘌呤结合口袋和DNA结合结构域。我们分别表达并纯化了人AGT的两个结构域。C-末端结构域在体外是完全无活性的，但通过冻融程序与N-末端结构域重组后获得了在Cys 145处形成S-烷基半胱氨酸的良好活性。这表明N-末端结构域在维持C-末端结构域的活性构型中起关键的结构作用。然而，当在缺乏内源性AGT的大肠杆菌细胞中表达时，该C-末端结构域单独具有保护免受甲基化剂N-甲基-N '-硝基-N-亚硝基胍（MNNG）的细胞毒性和诱变活性的活性，这表明其他蛋白质可以实现该功能。值得注意的是，游离的N-末端结构域的hAGT能够修复O-6-烷基鸟嘌呤在体外通过烷基转移，提供锌离子的存在。当在E.杆菌这种隐蔽的Zn ~（2+）依赖的DNA修复活性可能与AGTs的进化和功能有关。
O-6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects from alkylating agents by converting O-6-alkylguanine to guanine forming S-methylcysteine in the AGT protein. The crystal structure of human AGT shows clearly the presence of two domains. The N-terminal domain contains a bound zinc atom, and zinc binding confers a mechanistic enhancement to repair activity, but this domain has no known function. The C-terminal domain contains all residues so far implicated in alkyl transfer including the cysteine acceptor site (Cys145), the O-6-alkylguanine binding pocket, and a DNA binding domain. We have expressed and purified the two domains of human AGT separately. The C-terminal domain was totally inactive in vitro, but good activity forming S-alkylcysteine at Cys145 was obtained after recombination with the N-terminal domain via a freeze-thawing procedure. This suggests that the N-terminal domain plays a critical structural role in maintaining an active configuration of the C-terminal domain. However, this C-terminal domain alone had activity in protecting against the cytotoxic and mutagenic activity of the methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) when expressed in Escherichia coli cells lacking endogenous AGT, suggesting that other proteins can fulfill this function. Remarkably, the free N-terminal domain of hAGT was able to repair O-6-alkylguanine in vitro via alkyl transfer provided that zinc ions were present. The N-terminal domain was also able to produce moderate protection from MNNG when expressed in E. coli. This cryptic Zn2+-dependent DNA repair activity may be relevant to the evolution and function of AGTs.