Project 4 Gautier

摘要 DNA 双链断裂 (DSB) 修复对于防止持续性 DNA 损伤至关重要， 抵消基因组不稳定性并抑制肿瘤发展。空间组织 DNA 修复对于保护基因组免受染色体易位和复杂的影响至关重要 重新安排。因此，更好地理解其背后的机制至关重要 DNA 修复域的形成以及这些域对维持 基因组稳定性并最终抑制肿瘤。配备新颖的工具来操纵 为了形成同源定向修复域，我们提出了三个具体目标来评估 扰乱其形成对 DNA 修复和基因组稳定性的影响：突变的产生 和染色体重排。首先，我们将进一步研究肌动蛋白如何运动 WASP-ARP2/3 通路产生的参与 DSB 修复结构域的形成 WASP 中的 ARP2/3 抑制或临床相关突变。修复域将被探测 最先进的显微镜以及染色体构象捕获测定（Hi-C）。 接下来，我们将评估干扰 DNA 修复域形成对 基因组稳定性，特别是由各种基因组引起的染色体易位 侮辱：核酸内切酶、拓扑异构酶抑制剂和复制应激源。最后，我们将 使用损失和增益定义与 DSB 修复域缺陷相关的突变特征 WASP-ARP2/3 通路中的功能突变。该项目将极大受益 与该计划的所有其他项目的概念和技术互动。拟议的 实验应该提供对影响的前所未有的理解 细胞核的空间组织，即修复域，对基因组稳定性和后续 肿瘤的发展。

SUMMARY DNA double-strand break (DSB) repair is essential to prevent persistent DNA damage, counteract genome instability and suppress tumor development. The spatial organization of DNA repair is critical to protect the genome from chromosome translocations and complex rearrangements. Thus, it is critical to better understand the mechanisms underlying the formation of DNA repair domains and the contribution of these domains to the maintenance of genome stability and, ultimately, tumor suppression. Armed with novel tools to manipulate the formation of homology-directed repair domains, we propose three specific aims to assess the impact of perturbing their formation on DNA repair and genome stability: mutation generation and chromosome rearrangements. First, we will investigate further how actin movements generated by the WASP-ARP2/3 pathway participate in DSB repair domain formation using ARP2/3 inhibition or clinically relevant mutations in WASP. Repair domains will be probed by state-of-the-art microscopy as well as by chromosome conformation capture assays (Hi-C). Next, we will assess the consequences of interfering with DNA repair domain formation on genome stability, specifically on chromosome translocations arising from various genomic insults: endonucleases, topoisomerase inhibitors and replication stressors. Finally, we will define the mutational signatures associated with DSB repair domain defects using loss and gain of function mutations in the WASP-ARP2/3 pathway. This project will greatly benefit from conceptual and technical interactions with all other projects of the program. The proposed experiments should provide an unprecedented level of understanding of the impact of the spatial organization of the nucleus, i.e. repair domains, on genome stability and subsequent tumor development.