Department of Biochemistry, University of Colorado Boulder, Boulder, CO, 80309, USA.
Department of Biochemistry, University of Colorado Boulder, Boulder, CO, 80309, USA; Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, CO, 80309, USA.
DNA Repair (Amst). 2019 Sep;81:102650. doi: 10.1016/j.dnarep.2019.102650. Epub 2019 Jul 8.
All organisms must protect their genome from constantly occurring DNA damage. To this end, cells have evolved complex pathways for repairing sites of DNA lesions, and multiple in vitro and in vivo techniques have been developed to study these processes. In this review, we discuss the commonly used laser microirradiation method for monitoring the accumulation of repair proteins at DNA damage sites in cells, and we outline several strategies for deriving kinetic models from such experimental data. We discuss an example of how in vitro measurements and in vivo microirradation experiments complement each other to provide insight into the mechanism of PARP1 recruitment to DNA lesions. We also discuss a strategy to combine data obtained for the recruitment of many different proteins in a move toward fully quantitating the spatiotemporal relationships between various damage responses, and we outline potential venues for future development in the field.
所有生物都必须保护其基因组免受不断发生的 DNA 损伤。为此，细胞已经进化出了复杂的途径来修复 DNA 损伤部位，并且已经开发出了多种体外和体内技术来研究这些过程。在这篇综述中，我们讨论了常用的激光微照射方法，用于监测细胞中 DNA 损伤部位修复蛋白的积累，并概述了从这些实验数据中得出动力学模型的几种策略。我们讨论了一个例子，说明体外测量和体内微照射实验如何相互补充，以深入了解 PARP1 募集到 DNA 损伤部位的机制。我们还讨论了一种策略，即将许多不同蛋白质的募集数据结合起来，以全面量化各种损伤反应之间的时空关系，并概述了该领域未来发展的潜在途径。
