Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping
Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping
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DOI：
10.1093/nar/gku919
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发表时间：
2014-11-10
影响因子：
14.9
通讯作者：
O'Brien, Patrick J.
中科院分区：
文献类型：
--
作者：
Hendershot, Jenna M.;O'Brien, Patrick J.
核苷酸翻转是 DNA 修饰酶的一个常见特征，它允许进入双链 DNA 内的靶位点。结构研究已经在多种酶中鉴定出许多嵌入氨基酸侧链，但这些嵌入残基的功能贡献却知之甚少。我们使用定点诱变和瞬态动力学方法来剖析嵌入对人烷基腺嘌呤 DNA 糖基化酶（一种启动烷基化损伤修复的酶）的能量贡献。当 AAG 翻转出受损的核苷酸时，双链体中的空隙将被保守的酪氨酸 (Y162) 填充。我们发现酪氨酸嵌入使螺旋外特异性识别复合物稳定了 140 倍，并且 Y162 作为塞子发挥作用，相对于 Y162A 突变体，将展开速度减慢了 6000 倍。令人惊讶的是，相对于野生型酶，较小丙氨酸侧链的突变使核苷酸翻转速率增加了 50 倍。这提供了反对 DNA 嵌入加速核苷酸翻转这一流行模型的证据。就 AAG 而言，DNA 嵌入有助于受损核苷酸的特异性结合，但这种增强的特异性是以核苷酸翻转速度降低为代价的。
Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping.