Raina Vivek B, Greene Eric C
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA.
Methods Mol Biol. 2025;2881:195-211. doi: 10.1007/978-1-0716-4280-1_10.
Homologous recombination (HR) is the principal pathway undertaken by a cell for the error-free repair of DNA double-strand breaks that are frequently encountered by the cell. HR can be initiated at the sites of DNA double-strand breaks by generating long stretches of single-stranded 3' DNA overhang through a process called DNA end resection. In one DNA end resection pathway, this is achieved via the concerted effort of specialized machinery involving the RecQ family helicase BLM, the helicase/endonuclease DNA2, and a single-strand DNA binding protein complex RPA. BLM unwinds the DNA at the sites of DNA damage. The DNA unwound by BLM is cleaved in a 5' to 3' direction by DNA2 leaving behind a long single-stranded 3' DNA tail which is rapidly coated with RPA. This long single-stranded DNA provides the loading platform for recombinase proteins to form nucleoprotein filaments which initiate the homology search to undergo HR-based repair. Most of the insights into these processes have been gained using ensemble biochemical methods. However, these approaches have proven to be challenging for dissecting complex molecular mechanisms, especially because of the dynamic nature of these processes. Experiments involving single-molecule techniques have enabled us to counter these issues by allowing us to gather information on intermediates that are heterogeneous and transient in nature. We have developed a single-molecule technique called DNA curtains where we can study hundreds of protein-nucleic acid interaction events in real time. Here, we describe a method to prepare a single-tethered double-stranded DNA curtain to visualize, in combination with total internal reflection microscopy (TIRFM), the BLM- and DNA2-mediated DNA end resection in real time.
同源重组（HR）是细胞用于对DNA双链断裂进行无差错修复的主要途径，而DNA双链断裂在细胞中经常出现。HR可通过一种称为DNA末端切除的过程产生长链单链3' DNA突出端，从而在DNA双链断裂位点启动。在一种DNA末端切除途径中，这是通过涉及RecQ家族解旋酶BLM、解旋酶/核酸内切酶DNA2和单链DNA结合蛋白复合物RPA的专门机制协同作用来实现的。BLM在DNA损伤位点解开DNA。被BLM解开的DNA由DNA2以5'至3'方向切割，留下一条长的单链3' DNA尾巴，该尾巴迅速被RPA覆盖。这条长单链DNA为重组酶蛋白形成核蛋白丝提供了装载平台，核蛋白丝启动同源性搜索以进行基于HR的修复。对这些过程的大多数见解是通过整体生化方法获得的。然而，这些方法已被证明对于剖析复杂的分子机制具有挑战性，特别是由于这些过程的动态性质。涉及单分子技术的实验使我们能够通过收集关于本质上异质且短暂的中间体的信息来应对这些问题。我们开发了一种称为DNA帘的单分子技术，在该技术中我们可以实时研究数百个蛋白质 - 核酸相互作用事件。在这里，我们描述了一种制备单链双螺旋DNA帘的方法，以便结合全内反射显微镜（TIRFM）实时可视化BLM和DNA2介导的DNA末端切除。
