A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1
A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1
复制标题
DOI：
10.1074/jbc.m509137200
复制
发表时间：
2006-02-24
影响因子：
4.8
通讯作者：
Baldwin, GS
中科院分区：
文献类型：
--
作者：
Krusong, K;Carpenter, EP;Baldwin, GS
尿嘧啶-DNA糖基化酶（UNG）是启动碱基切除修复的关键酶。我们已经使用动力学和结合试验对来自人类和单纯疱疹病毒1型（HSV-1）的UNG酶进行了比较分析。稳态荧光分析表明，由于Km较低，与病毒酶相比，hUNG具有高得多的特异性常数（k（cat）/K-m）。还使用UNG的催化失活突变体和不可切割的底物类似物（2 '-脱氧尿苷和2'-α-氟-2 '-脱氧尿苷）研究了UNG与DNA的结合。平衡DNA结合显示，人类和HSV-1 UNG酶与脱碱基DNA和两种底物类似物的结合比与含尿嘧啶的DNA的结合更弱。与尿嘧啶复合的HSV-1 D88 N/H210 N UNG的结构测定揭示了底物结合的详细信息。总之，这些结果表明，结合能的一个显着的比例是由与目标尿嘧啶的特异性相互作用。人UNG的动力学参数表明，它可能对U（.）A和U（.）体内G不匹配。与脱碱基DNA的弱结合也表明UNG活性不太可能与随后的碱基切除修复的常见步骤偶联。
Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K-m) compared with the viral enzyme due to a lower Km. The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U(.)A and U(.)G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.