Systematic analysis of the molecular and biophysical properties of key DNA damage response factors.
Systematic analysis of the molecular and biophysical properties of key DNA damage response factors.
复制标题
DOI：
10.7554/elife.87086
复制
发表时间：
2023-06-21
期刊：
影响因子：
7.7
通讯作者：
Schmidt JC
中科院分区：
文献类型：
--
作者：
Heyza JR;Mikhova M;Bahl A;Broadbent DG;Schmidt JC
DNA双链断裂(DSB)的修复是保持基因组完整性所不可或缺的。因此，明确DSB修复的机制将增强我们对这些途径中的缺陷如何导致人类疾病的理解，并可能导致发现新的治疗干预方法。在这里，我们在U2OS细胞中建立了一组HaloTag标记的DNA损伤反应因子，使荧光HaloTag配体能够进行浓度依赖的蛋白质标记。将HaloTag插入到这些修复因子的内源基因座上，可以保持表达水平，蛋白质保持适当的亚细胞定位、病灶形成能力，并在功能上支持DSB修复。我们系统地分析了细胞总蛋白丰度，测量了激光诱导DNA损伤部位的募集动力学，并通过活细胞单分子成像确定了扩散动力学和染色质结合特性。我们的工作表明，Shieldin复合体作为末端连接的关键因子，并不以预组装状态存在，并且这些因子在DSB上的相对积累以不同的动力学方式发生。此外，活细胞单分子成像揭示了MDC1与染色质之间由其PST重复结构域介导的结构性相互作用。综上所述，我们的研究证明了单分子成像为DNA修复提供了机械性的见解，这将成为表征活细胞中DNA修复因子的生物物理性质的强大资源。
Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling by fluorescent HaloTag ligands. Genomic insertion of HaloTag at the endogenous loci of these repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to laser-induced DNA damage sites, and defined the diffusion dynamics and chromatin binding characteristics by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end-joining, does not exist in a preassembled state and that relative accumulation of these factors at DSBs occurs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by its PST repeat domain. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.