Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Korea.
Department of Bio-Analytical Science, University of Science & Technology, Daejeon, Korea.
Photochem Photobiol. 2017 Jan;93(1):192-198. doi: 10.1111/php.12638. Epub 2016 Nov 3.
The human nucleotide excision repair system targets a wide variety of DNA adducts for removal from DNA, including photoproducts induced by UV wavelengths of sunlight. A key feature of nucleotide excision repair is its dual incision mechanism, which results in generation of a small, damage-containing oligonucleotide approximately 24 to 32 nt in length. Detection of these excised oligonucleotides using cell-free extracts and purified proteins with defined DNA substrates has provided a robust biochemical assay for excision repair activity in vitro. However, the relevance of a number of in vitro findings to excision repair in living cells in vivo has remained unresolved. Over the past few years, novel methods for detecting and isolating the excised oligonucleotide products of repair in vivo have therefore been developed. Here we provide a basic outline of a sensitive and versatile in vivo excision assay and discuss how the assay both confirms previous in vitro findings and offers a number of advantages over existing cell-based DNA repair assays. Thus, the in vivo excision assay offers a powerful tool for readily monitoring the repair of DNA lesions induced by a large number of environmental carcinogens and anticancer compounds.
人类核苷酸切除修复系统针对 DNA 中的多种 DNA 加合物进行去除，包括由太阳光中紫外线波长诱导的光产物。核苷酸切除修复的一个关键特征是其双切割机制，该机制导致生成一个小的、含有损伤的寡核苷酸，其长度约为 24 至 32 个核苷酸。使用无细胞提取物和具有定义 DNA 底物的纯化蛋白检测这些切除的寡核苷酸，为体外切除修复活性提供了一种强大的生化测定方法。然而，许多体外发现与体内活细胞中的切除修复的相关性仍未得到解决。在过去的几年中，因此开发了用于检测和分离体内修复切除寡核苷酸产物的新方法。在这里，我们提供了一种灵敏且通用的体内切除测定的基本概述，并讨论了该测定如何既证实了先前的体外发现，又为现有的基于细胞的 DNA 修复测定提供了许多优势。因此，体内切除测定为监测由大量环境致癌剂和抗癌化合物诱导的 DNA 损伤的修复提供了一种强大的工具。
