Kong Xiangduo, Cruz Gladys M S, Silva Bárbara A, Wakida Nicole M, Khatibzadeh Nima, Berns Michael W, Yokomori Kyoko
Department of Biological Chemistry, School of Medicine, University of California, Irvine.
Beckman Laser Institute and Medical Clinic, University of California, Irvine.
J Vis Exp. 2018 Jan 31(131):56213. doi: 10.3791/56213.
DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.
DNA损伤会在细胞中引发特定的信号传导和修复反应，这对于保护基因组完整性至关重要。激光微照射成为研究体内DNA损伤反应（DDR）的一种有价值的实验工具。它能够对局限于细胞核亚微米区域的激光诱导损伤做出反应的大分子动力学进行实时高分辨率单细胞分析。然而，人们在使用各种激光条件时并未意识到所诱导损伤类型的差异。结果，损伤的性质往往没有得到很好的表征或控制，导致在招募或修饰图谱中出现明显的不一致。我们证明，不同的照射条件（即飞秒（fs）近红外（NIR）激光的不同波长以及不同输入功率（辐照度））会诱导不同的DDR和修复蛋白组装。这反映了所产生的DNA损伤类型。本方案描述了如何通过滴定激光输入功率来诱导不同数量和复杂性的DNA损伤，通过检测碱基和交联损伤、差异多聚（ADP-核糖）（PAR）信号传导以及损伤位点的途径特异性修复因子组装，这些损伤很容易被监测到。一旦确定了损伤条件，就有可能研究不同损伤复杂性和差异损伤信号传导以及上游因子缺失对任何感兴趣因子的影响。
