Subramanyam Shyamal, Spies Maria
University of Iowa Carver College of Medicine, Iowa City, IA, United States.
University of Iowa Carver College of Medicine, Iowa City, IA, United States.
Methods Enzymol. 2018;600:157-178. doi: 10.1016/bs.mie.2017.11.011. Epub 2018 Jan 9.
The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to "rogue," genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cancerous cells. Human RAD51 is, therefore, an important and highly coveted anticancer drug target. Biochemical, biophysical, and structural studies of the human RAD51 and establishment of its structure-activity relationship require purification of large quantities of protein. In this chapter we describe a robust method for expression and purification of human RAD51 and the methods for assessing its activity based on the single-strand DNA-binding stoichiometry and its capacity to carry out the DNA strand exchange reaction.
RAD51 DNA链交换蛋白在维持人类基因组完整性方面发挥着重要作用。它通过在受损DNA分子和完整DNA分子之间交换链来促进同源性定向DNA修复。它在稳定受损的DNA复制叉方面也起着重要作用。然而，当RAD51过度表达或调控异常时，它会导致“异常”的基因组不稳定事件，进而引发癌症、细胞死亡以及癌细胞获得化疗耐药性。因此，人类RAD51是一个重要且备受关注的抗癌药物靶点。对人类RAD51进行生化、生物物理和结构研究并建立其构效关系需要大量蛋白质的纯化。在本章中，我们描述了一种高效表达和纯化人类RAD51的方法，以及基于单链DNA结合化学计量和进行DNA链交换反应能力来评估其活性的方法。
