Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes
Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes
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DOI：
10.1016/j.ab.2007.04.049
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发表时间：
2007-07-15
影响因子：
2.9
通讯作者：
Tang, Hongxing
中科院分区：
文献类型：
--
作者：
Liu, Bin;Yang, Xiaohai;Tang, Hongxing
作为高度保守的损伤修复蛋白，尿嘧啶-DNA糖基化酶（UDG）主要催化尿嘧啶与DNA的切除，以维持基因组完整性。在这里，引入了一种实时监测尿嘧啶去除的新方法。用尿嘧啶残基修饰的双链DNA探针在荧光共振能量转移（FRET）中被用作底物，并检测均匀溶液中的探针。这种方法不仅克服了传统放射性测定的缺点，例如不连续性和耗时且复杂，而且还用于准确确定UDG的动力学常数。 UDG的检测极限为0.033 U/ml。 K-M和K-CAT分别为0.11 mu m和4 s（-1）。此外，该方法用于研究化学药物对UDG活性的影响。结果表明，氟尿嘧啶（5-FU）和庆大霉素是UDG的抑制剂。 A549细胞中UDG的体外检测表明，用顺铂处理细胞后UDG的活性高四倍。这些结果表明，该方法可以实时监测尿嘧啶的去除，并方便地测定UDG活性，具有超敏性和出色的均匀溶液特异性。该方法还可以在体外进行高通量药物筛查。 （c）2007 Elsevier Inc.保留所有权利。
As a highly conserved damage repair protein, uracil-DNA glycosylase (UDG) mainly catalyzes the excision of uracil from DNA to sustain the genome integrity. Here a novel method for monitoring the uracil removal in real time is introduced. Double-stranded DNA probes modified with uracil residues that can occur in fluorescent resonance energy transfer (FRET) were used as substrates and detecting probes in a homogeneous solution. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also was used to accurately determine the kinetic constant of UDG. The limit of detection of UDG was 0.033 U/ml. The K-m and K-cat were 0.11 mu M and 4 s(-1), respectively. In addition, the method was applied to investigate the influence of chemical drugs on UDG activity. The results showed that 10 mM fluorouracil (5-FU) and gentamicin are inhibitors to UDG. The in vitro detection of UDG in A549 cells showed that the activity of UDG was four times greater after the cells were treated with cisplatin. These results showed that this method can monitor uracil removal in real time and conveniently assay UDG activity with ultrasensitivity and excellent specificity in the homogeneous solution. This method is also amenable to high-throughput drug screening in vitro. (c) 2007 Elsevier Inc. All rights reserved.