Wang Lan, Lee Seung-Joo, Verdine Gregory L
From the Departments of Chemistry and Chemical Biology.
From the Departments of Chemistry and Chemical Biology, Stem Cell and Regenerative Biology, and Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
J Biol Chem. 2015 Jul 10;290(28):17096-105. doi: 10.1074/jbc.M115.657866. Epub 2015 May 20.
The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site.
DNA中具有高度致突变性的A:氧代鸟嘌呤（8-氧代鸟嘌呤）碱基对最常由初级氧化损伤C:氧代鸟嘌呤的异常复制产生。这种损伤特别隐匿，因为其组成的两个核碱基都不能忠实地从原始的C:G碱基对传递遗传信息。A:氧代鸟嘌呤的修复由腺嘌呤DNA糖基化酶启动，该酶催化从DNA主链上水解切割异常的A核碱基。这些酶，细菌中的MutY和人类中的MUTYH，会严格避免处理C:氧代鸟嘌呤，因为切割C:氧代鸟嘌呤中的C残基实际上会促进向A:氧代鸟嘌呤的致突变转化。在这里，我们使用合成晶体学方法分析MutY排斥C:氧代鸟嘌呤的结构基础，以捕获该酶在检查C:氧代鸟嘌呤反底物的过程中，该酶通常仅短暂结合该反底物。我们发现MutY使用两种不同的策略来避免将C呈递给酶活性位点。首先，MutY拥有一个外位点作为C的诱饵，其次，与关键活性位点残基的排斥力阻止C稳定插入酶活性位点内的核碱基识别口袋。
