Coordinating the Initial Steps of Base Excision Repair APURINIC/APYRIMIDINIC ENDONUCLEASE 1 ACTIVELY STIMULATES THYMINE DNA GLYCOSYLASE BY DISRUPTING THE PRODUCT COMPLEX
Coordinating the Initial Steps of Base Excision Repair APURINIC/APYRIMIDINIC ENDONUCLEASE 1 ACTIVELY STIMULATES THYMINE DNA GLYCOSYLASE BY DISRUPTING THE PRODUCT COMPLEX
复制标题
DOI：
10.1074/jbc.m805504200
复制
发表时间：
2008-11-21
影响因子：
4.8
通讯作者：
Drohat, Alexander C.
中科院分区：
文献类型：
--
作者：
Fitzgerald, Megan E.;Drohat, Alexander C.
DNA 糖基化酶通过去除受损或不匹配的碱基来启动碱基切除修复，产生无嘌呤/无嘧啶 (AP) DNA。对于许多糖基化酶来说，AP-DNA 保持紧密结合，阻碍酶的周转。一个突出的例子是胸腺嘧啶 DNA 糖基化酶 (TDG)，它可以从 G.T 错配中去除 T，并识别其他病变，对 CpG 二核苷酸的损伤具有特异性。 TDG周转很慢；当[产物]/[酶]比例接近统一时，其活性似乎达到稳定水平。后续碱基切除修复酶 AP 核酸内切酶 1 (APE1) 会刺激 TDG 和其他糖基酶的周转，其机制目前尚不清楚。我们使用前稳态动力学和偶联酶 (hTDG-hAPE1) 荧光测定法检测了人 TDG (hTDG) 单独以及与人 APE1 (hAPE1) 的催化活性。对于 GT (k(cat) = 0.00034 min(-1)) 和 G.U (k(cat) = 0.005 min(-1)) 底物，hTDG 周转极其缓慢，比单次周转实验中的 k(max) 慢得多，这证实了 AP-DNA 释放是限速的。我们发现 G.T、G.U 和 G.FU 底物的 kcat 存在出乎意料的巨大差异，表明切除的碱基仍然被 AP-DNA 捕获在产物复合物中。 hAPE1 使 G.T 和 G.U 底物的 hTDG 周转率增加 42 倍和 26 倍，这是 hAPE1 作用的第一个定量测量。 hAPE1 通过破坏产物复合物而不是仅仅消耗（核酸内切）AP-DNA 来刺激 hTDG。 hTDG 催化核心（410 的残基 111-308）的增强更大，表明 N 端和 C 端结构域对于与 hAPE1 的刺激相互作用是可有可无的。讨论了 hAPE1 破坏 hTDG 产物复合物的潜在机制。
DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing apurinic/apyrimidinic (AP) DNA. For many glycosylases, the AP-DNA remains tightly bound, impeding enzymatic turnover. A prominent example is thymine DNA glycosylase (TDG), which removes T from G.T mispairs and recognizes other lesions, with specificity for damage at CpG dinucleotides. TDG turnover is very slow; its activity appears to reach a plateau as the [product]/[enzyme] ratio approaches unity. The follow-on base excision repair enzyme, AP endonuclease 1 (APE1), stimulates the turnover of TDG and other glycosylases, involving a mechanism that remains largely unknown. We examined the catalytic activity of human TDG (hTDG), alone and with human APE1 (hAPE1), using pre-steady-state kinetics and a coupled-enzyme (hTDG-hAPE1) fluorescence assay. hTDG turnover is exceedingly slow for G.T (k(cat) = 0.00034 min(-1)) and G.U (k(cat) = 0.005 min(-1)) substrates, much slower than k(max) from single turnover experiments, confirming that AP-DNA release is rate-limiting. We find unexpectedly large differences in kcat for G.T, G.U, and G.FU substrates, indicating the excised base remains trapped in the product complex by AP-DNA. hAPE1 increases hTDG turnover by 42- and 26-fold for G.T and G.U substrates, the first quantitative measure of the effect of hAPE1. hAPE1 stimulates hTDG by disrupting the product complex rather than merely depleting (endonucleolytically) the AP-DNA. The enhancement is greater for hTDG catalytic core (residues 111-308 of 410), indicating the N- and C-terminal domains are dispensable for stimulatory interactions with hAPE1. Potential mechanisms for hAPE1 disruption of the of hTDG product complex are discussed.