Institute of Biochemistry, Justus-Liebig University, Giessen, Germany.
Biotechniques. 2011 Oct;51(4):259-62, 264-5. doi: 10.2144/000113749.
The DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current approaches used to generate MMR substrates are time-consuming and/or not very flexible with respect to sequence context. Here we report an approach to generate small circular DNA containing a mismatch (nanocircles). Our method is based on the nicking of PCR products resulting in single-stranded 3' overhangs, which form DNA circles after annealing and ligation. Depending on the DNA template, one can generate mismatched circles containing a single hemimethylated GATC site (for use with the bacterial system) and/or nicking sites to generate DNA circles nicked in the top or bottom strand (for assays with the bacterial or eukaryotic MMR system). The size of the circles varied (323 to 1100 bp), their sequence was determined by the template DNA, and purification of the circles was achieved by ExoI/ExoIII digestion and/or gel extraction. The quality of the nanocircles was assessed by scanning-force microscopy and their suitability for in vitro repair initiation was examined using recombinant Escherichia coli MMR proteins.
DNA 错配修复 (MMR) 系统识别和修复逃脱 DNA 聚合酶校对功能的错误。为了研究 MMR 机制的分子细节，体外生化测定需要携带错配和链分辨信号的特定 DNA 底物。目前用于生成 MMR 底物的方法既耗时又/或对序列背景的适应性不强。在这里，我们报告了一种生成含有错配的小圆形 DNA（纳米环）的方法。我们的方法基于 PCR 产物的切口，导致单链 3'突出，在退火和连接后形成 DNA 环。根据 DNA 模板，可以生成含有单个半甲基化 GATC 位点的错配环（用于细菌系统）和/或切口位点，以生成在顶部或底部链上切口的 DNA 环（用于细菌或真核 MMR 系统的测定）。环的大小不同（323 到 1100 bp），其序列由模板 DNA 决定，通过 ExoI/ExoIII 消化和/或凝胶提取来纯化环。通过扫描力显微镜评估纳米环的质量，并使用重组大肠杆菌 MMR 蛋白检查它们在体外修复起始中的适用性。
