Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability
Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability
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DOI：
10.1021/ja0634829
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发表时间：
2006-09-27
影响因子：
15
通讯作者：
Drohat, Alexander C.
中科院分区：
文献类型：
--
作者：
Bennett, Matthew T.;Rodgers, M. T.;Drohat, Alexander C.
DNA 糖基化酶启动 DNA 碱基切除修复途径，发现并水解切除 DNA 中受损的碱基。虽然一些 DNA 糖基化酶表现出狭窄的特异性，但其他糖基化酶可以消除多种形式的损伤。人胸腺嘧啶 DNA 糖基化酶 (hTDG) 将胸腺嘧啶从诱变的 G 中心点 T 错配中裂解出来，识别许多其他损伤，并且对与鸟嘌呤而不是腺嘌呤配对的核碱基具有强烈的偏好。然而，hTDG 避免了胞嘧啶，尽管正常 G 中心点 C 对比 G、T 错配多出数百万倍。这种显着而本质的特异性的机制仍然不清楚。在这里，我们通过确定针对一系列具有不同离去基团能力的核碱基的最大活性 (k(max)) 来检查 hTDG 特异性取决于易裂碱基糖键稳定性的可能性。我们发现 hTDG 去除 5-氟尿嘧啶的速度比尿嘧啶快 78 倍，去除 5-氯尿嘧啶的速度比胸腺嘧啶快 572 倍，这些差异主要归因于离去基团能力。此外，hTDG 可以轻松切除胞嘧啶类似物，并具有改善的离开能力，包括 5-氟胞嘧啶、5-溴胞嘧啶和 5-羟基胞嘧啶，表明胞嘧啶可以进入活性位点。 log(k(max)) 与离去基团 pK(a) 的关系图揭示了布朗斯台德型线性自由能关系，具有大的负斜率 beta lg = -1.6 +/- 0.2，与高度解离反应机制一致。此外，我们发现 hTDG 的疏水活性位点通过增强底物反应性的固有差异而有助于其特异性。因此，hTDG 特异性取决于 N-糖苷键稳定性，而对胞嘧啶的歧视很大程度上是由于其非常差的离开能力而不是其被排除在活性位点之外。
Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G center dot T mispairs, recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the million-fold excess of normal G center dot C pairs over G, T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bond by determining the maximal activity (k(max)) against a series of nucleobases with varying leaving-group ability. We find that hTDG removes 5-fluorouracil 78-fold faster than uracil, and 5-chlorouracil, 572-fold faster than thymine, differences that can be attributed predominantly to leaving-group ability. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k(max)) versus leaving-group pK(a) reveals a Bronsted-type linear free energy relationship with a large negative slope of beta lg = -1.6 +/- 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.