Krosky Daniel J, Schwarz Frederick P, Stivers James T
Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Biochemistry. 2004 Apr 13;43(14):4188-95. doi: 10.1021/bi036303y.
To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.
为了有效地维持其基因组完整性，DNA修复糖基化酶必须利用一种螺旋外识别机制，对其同源的受损碱基表现出高催化特异性。对特异性的一种可能贡献是受损位点的弱碱基配对和固有不稳定性，这可能导致受损碱基的螺旋外度增加，并增强对这些位点的识别。该模型预测，随着损伤碱基对的热力学稳定性降低，酶的结合亲和力应该增加，因为将碱基挤出到其活性位点所需的功更少。我们通过构建一系列DNA双链体来测试尿嘧啶DNA糖基化酶（UDG）的这一假设，这些双链体包含一个与各种碱基（X）相对的单个尿嘧啶（U），X与U形成零至三个氢键。观察到线性自由能（LFE）关系，其将UDG结合亲和力与双链体解链的熵和焓以及受损位点对化学氧化的动态可及性相关联。这些LFE表明，碱基对的焓不稳定导致受损位点构象自由度增加，促进了螺旋外状态的形成，从而将特异性识别提高了多达3000倍。然而，鉴于正常碱基对与U·A或U·G碱基对之间的稳定性差异较小，相对碱基对稳定性对UDG在细胞DNA中对尿嘧啶位点>10^6倍的区分贡献不大。相比之下，其他更严重的DNA损伤的固有不稳定性可能对与螺旋外碱基结合的其他DNA修复酶的特异性有显著贡献。
