Brown M W, de la Torre A, Finkelstein I J
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States.
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, United States.
Methods Enzymol. 2017;582:221-238. doi: 10.1016/bs.mie.2016.08.006. Epub 2016 Oct 24.
The DNA mismatch repair (MMR) system corrects errors that occur during DNA replication. MMR needs the coordinated and highly dynamic assembly of repair enzymes at the site of the lesion. By visualizing transient intermediates of these assemblies, single-molecule approaches have shed critical insights into the mechanisms of MMR. These studies frequently require long (>20kb) DNA substrates with lesions and other extrahelical structures inserted at defined positions. DNA derived from bacteriophage λ (λ-DNA) is a high quality long (48.5kb) DNA substrate that is frequently used in single-molecule studies. Here we provide detailed protocols for site-specific incorporation of recombinant sequences and extrahelical structures into λ-DNA. We also describe how to assemble DNA curtains, and how to collect and analyze single-molecule observations of lesion recognition by MMR proteins diffusing on these DNA curtains. These protocols will facilitate future single-molecule studies of DNA transcription, replication, and repair.
DNA错配修复（MMR）系统可纠正DNA复制过程中出现的错误。MMR需要修复酶在损伤位点进行协调且高度动态的组装。通过可视化这些组装的瞬时中间体，单分子方法为MMR机制提供了关键见解。这些研究通常需要长（>20kb）的DNA底物，其中损伤和其他螺旋外结构插入到特定位置。源自噬菌体λ（λ-DNA）的DNA是一种高质量的长（48.5kb）DNA底物，常用于单分子研究。在这里，我们提供了将重组序列和螺旋外结构位点特异性掺入λ-DNA的详细方案。我们还描述了如何组装DNA帘幕，以及如何收集和分析在这些DNA帘幕上扩散的MMR蛋白对损伤识别的单分子观察结果。这些方案将促进未来对DNA转录、复制和修复的单分子研究。
