Project 4 Gautier

摘要 DNA双链断裂(DSB)修复是防止持续DNA损伤的关键， 中和基因组不稳定，抑制肿瘤发展。城市的空间组织 DNA修复是保护基因组免受染色体易位和复杂性的关键 重新安排。因此，更好地了解 DNA修复结构域的形成及其在维持DNA损伤中的作用 基因组的稳定性，以及最终的肿瘤抑制。配备了新的工具来操纵 同源定向修复结构域的形成，我们提出了三个具体的目标来评估 干扰它们的形成对DNA修复和基因组稳定性的影响：突变产生 和染色体重排。首先，我们将进一步研究肌动蛋白是如何移动的 由WASP-Arp2/3途径产生参与DSB修复结构域的形成 WASP的Arp2/3抑制或临床相关突变。修复域将通过以下方式进行探测 最先进的显微镜以及染色体构象捕获分析(Hi-C)。 接下来，我们将评估干扰DNA修复结构域形成的后果 基因组稳定性，特别是不同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，我们会 使用损失和增益定义与DSB修复结构域缺陷相关的突变特征 WASP-Arp2/3途径的功能突变。这个项目将极大地受益于 与项目的所有其他项目进行概念和技术上的互动。建议数 实验应该提供一个史无前例的水平来理解 核的空间组织，即修复结构域，对基因组稳定性和随后的 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.