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使用荧光共振能量转移探针实时监测尿嘧啶-DNA糖基化酶去除尿嘧啶的过程。

Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes.

作者信息

Liu Bin, Yang Xiaohai, Wang Kemin, Tan Weihong, Li Huimin, Tang Hongxing

机构信息

Institute of Life Science and Biotechnology, College of Chemistry and Chemical Engineering, Hunan University, and Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, People's Republic of China.

出版信息

Anal Biochem. 2007 Jul 15;366(2):237-43. doi: 10.1016/j.ab.2007.04.049. Epub 2007 May 3.

Abstract

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 KM and Kcat were 0.11 microM 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.

摘要

作为一种高度保守的损伤修复蛋白,尿嘧啶-DNA糖基化酶(UDG)主要催化从DNA中切除尿嘧啶以维持基因组完整性。本文介绍了一种实时监测尿嘧啶去除的新方法。用可发生荧光共振能量转移(FRET)的尿嘧啶残基修饰的双链DNA探针在均相溶液中用作底物和检测探针。该方法不仅克服了传统放射性检测方法的缺点,如不连续性、耗时和复杂,还用于准确测定UDG的动力学常数。UDG的检测限为0.033 U/ml。KM和Kcat分别为0.11 microM和4 s(-1)。此外,该方法还用于研究化学药物对UDG活性的影响。结果表明,10 mM氟尿嘧啶(5-FU)和庆大霉素是UDG的抑制剂。对A549细胞中UDG的体外检测表明,用顺铂处理细胞后,UDG的活性提高了四倍。这些结果表明,该方法可以实时监测尿嘧啶的去除,并在均相溶液中以超灵敏度和优异的特异性方便地测定UDG活性。该方法也适用于体外高通量药物筛选。

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