在DNA帘幕上组装人类切除体。
Assembling the Human Resectosome on DNA Curtains.
作者信息
Soniat Michael M, Myler Logan R, Finkelstein Ilya J
机构信息
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA.
Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, USA.
出版信息
Methods Mol Biol. 2019;1999:225-244. doi: 10.1007/978-1-4939-9500-4_14.
DNA double-strand breaks (DSBs) are a potentially lethal DNA lesions that disrupt both the physical and genetic continuity of the DNA duplex. Homologous recombination (HR) is a universally conserved genome maintenance pathway that initiates via nucleolytic processing of the broken DNA ends (resection). Eukaryotic DNA resection is catalyzed by the resectosome-a multicomponent molecular machine consisting of the nucleases DNA2 or Exonuclease 1 (EXO1), Bloom's helicase (BLM), the MRE11-RAD50-NBS1 (MRN) complex, and additional regulatory factors. Here, we describe methods for purification and single-molecule imaging and analysis of EXO1, DNA2, and BLM. We also describe how to adapt resection assays to the high-throughput single-molecule DNA curtain assay. By organizing hundreds of individual molecules on the surface of a microfluidic flowcell, DNA curtains visualize protein complexes with the required spatial and temporal resolution to resolve the molecular choreography during critical DNA-processing reactions.
DNA双链断裂（DSB）是一种潜在的致死性DNA损伤，它会破坏DNA双链的物理和遗传连续性。同源重组（HR）是一种普遍保守的基因组维持途径，通过对断裂的DNA末端进行核酸酶处理（切除）来启动。真核生物的DNA切除由切除体催化，切除体是一种多组分分子机器，由核酸酶DNA2或核酸外切酶1（EXO1）、布鲁姆解旋酶（BLM）、MRE11-RAD50-NBS1（MRN）复合物以及其他调节因子组成。在这里，我们描述了EXO1、DNA2和BLM的纯化、单分子成像及分析方法。我们还描述了如何将切除分析方法应用于高通量单分子DNA幕帘分析。通过在微流控流动池表面组织数百个单个分子，DNA幕帘以所需的空间和时间分辨率可视化蛋白质复合物，从而解析关键DNA处理反应过程中的分子编排。
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