Sidhu Arshdeep, Ristic Dejan, Sánchez Humberto, Wyman Claire
Erasmus University Medical Center, Rotterdam, The Netherlands.
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
Methods Enzymol. 2018;600:347-374. doi: 10.1016/bs.mie.2017.11.014.
Cellular functions are defined by dynamic assembly, rearrangement, and disassembly of biomolecules to achieve control and specificity. As an example, effective DNA repair is brought about by the concerted action of several DNA processing proteins. Both changes in the structure of individual proteins and in the arrangement of multiple proteins together (referred to here as architecture) are inherent to biological function. These dynamic changes are exemplified in the breast cancer susceptibility protein 2 (BRCA2). BRCA2 is a DNA repair protein that undergoes changes in its own structure and affects changes in molecular architecture with partners during homologous recombination (HR) repair of DNA double strand breaks. These challenging features of BRCA2 protein, its size and predicted stretches of intrinsically disordered regions, have made it difficult to determine the structural consequences and mechanistic importance of interactions between full-length BRCA2 with RAD51 and other HR proteins. In this chapter, we describe scanning force microscopy (SFM)-based approaches to study DNA-protein complexes involved in HR, the architectural plasticity of full-length BRCA2, and the dynamic reorganization of these molecular components associated with essential steps of HR.
细胞功能是由生物分子的动态组装、重排和解聚来实现控制和特异性的。例如，有效的DNA修复是由几种DNA加工蛋白的协同作用实现的。单个蛋白质结构的变化以及多种蛋白质共同排列方式（这里称为结构）的变化都是生物学功能所固有的。这些动态变化在乳腺癌易感蛋白2（BRCA2）中得到体现。BRCA2是一种DNA修复蛋白，在DNA双链断裂的同源重组（HR）修复过程中，它自身结构会发生变化，并影响与其伙伴的分子结构变化。BRCA2蛋白的这些具有挑战性的特征、其大小以及预测的内在无序区域片段，使得确定全长BRCA2与RAD51及其他HR蛋白之间相互作用的结构后果和机制重要性变得困难。在本章中，我们描述了基于扫描力显微镜（SFM）的方法来研究参与HR的DNA-蛋白质复合物、全长BRCA2的结构可塑性以及与HR基本步骤相关的这些分子成分的动态重组。
