Homologous Recombination Repair Domains: Formation and Impact on Genome Stability

同源重组修复域：形成及其对基因组稳定性的影响

• 批准号: 10440346
• 负责人: Jennifer Ashley Zagelbaum
• 金额: $ 5.18万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10440346
• 关键词: ANGPTL2 gene; Actins; Affect; BRCA1 gene; BRCA2 gene; Biological Assay; Cell Cycle; Cell Cycle Stage; Cell Line; Cells; Chemotherapy-Oncologic Procedure; Chromosomal Rearrangement; Chromosomal translocation; Complement; Complex; DNA; DNA Adducts; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Restriction Enzymes; Data; Defect; Development; Double Strand Break Repair; Down-Regulation; Etoposide; Event; Excision; Fibrinogen; Filament; Frequencies; Generations; Genes; Genetic; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Hi-C; Individual; Knock-out; Lead; Link; Location; Malignant Neoplasms; Mammalian Cell; Mediating; Movement; Mutate; Mutation; Nonhomologous DNA End Joining; Nuclear; Pathologic; Pathway interactions; Phosphotransferases; Play; Poisoning; Polymers; Process; Proteins; Role; Site; Small Interfering RNA; Technology; Therapy-Related Acute Myeloid Leukemia; Topoisomerase II; Topoisomerase-II Inhibitor; Tumor Suppressor Genes; WASP protein; Wiskott-Aldrich Syndrome; Work; Yeasts; alpha-Thalassemia; carcinogenesis; chemotherapeutic agent; chromosome conformation capture; experimental study; gene interaction; gene translocation; genome-wide; genome-wide analysis; genomic locus; genotoxicity; homologous recombination; improved mobility; inhibitor; insight; leukemia; live cell imaging; novel; polymerization; prevent; recombinational repair; repaired; small molecule inhibitor; soft tissue; tumor; tumorigenesis

PROJECT SUMMARY: DNA double-strand break (DSB) repair is spatially organized into nuclear repair domains that specifically facilitate DSB repair by homologous recombination (HR). HR, one of the major DSB pathways along with non-homologous end-joining, has been implicated in tumorigenesis, notably following mutations in the tumor suppressor genes BRCA1 and BRCA2 [1, 2]. Our lab demonstrated that upon DSB formation by induction of a restriction endonuclease (RE) or treatment with neocarzinostatin (NCS), WASP activates ARP2/3, which polymerizes nuclear actin into branched filaments [4]. This enhances the mobility of DSBs destined for HR and their subsequent clustering into HR domains. The DNA topoisomerase II (Top2) inhibitor etoposide (ETO) yields DSBs harboring protein-DNA adducts that require resection and subsequent repair by HR factors, including MRN, CtIP, and BRCA1 [5, 6]. Because of the absolute requirement for poisoned Top2 removal prior to repair, ETO is a unique way to probe the functional relationship between resection and movement. ETO is used to treat a wide range of cancers, including leukemia and soft tissue cancers. However, treatment is associated with secondary leukemias due to translocations. Using live-cell imaging, I show that ETO DSBs undergo ARP2/3-mediated movement and clustering. However, unlike RE and NCS DSBs, movement is not restricted to G2 but also occurs in G1. Additionally, ETO breaks in G1 undergo resection and load HR machinery, such as RPA. I have also begun examining the role of HR factors, including Mre11 and BRCA2, in repair domain formation following the generation of DSBs by RE, NCS and ETO. Although DSB clustering is crucial for HR, little is known about how repair domains are formed and their local and genome-wide implications. For example, we do not fully understand the crosstalk between movement (actin, WASP) and repair (HR machinery) in mammalian cells. Additionally, the dynamics of DSBs likely influences chromosomal rearrangements. Our lab is integrating high-throughput genomic technologies that assess gene- gene interactions and translocation events to determine the genome-wide implications of DSB mobility. The overarching goals of this study are to elucidate mechanisms by which nuclear actin polymerization and HR proteins regulate repair domain formation and to evaluate the genome-wide impact of DSB mobility. I hypothesize that HR proteins, including the resection machinery, play a critical role in regulating ARP2/3- mediated DSB movements and subsequent clustering. I further propose that nuclear actin polymerization impacts genome organization following DNA damage and thus affects translocation frequency. I will investigate these hypotheses in the following aims: Aim 1: Elucidate the contribution of HR machinery to Arp2/3-dependent DSB clustering. Aim 2: Determine the impact of ARP2/3-mediated DSB movement on genome stability.

项目摘要：DNA 双链断裂 (DSB) 修复在空间上组织成核修复域 其通过同源重组 (HR) 专门促进 DSB 修复。 HR，DSB 的主要途径之一 与非同源末端连接一起，与肿瘤发生有关，特别是在突变之后 抑癌基因 BRCA1 和 BRCA2 [1, 2]。我们的实验室证明 DSB 形成后 诱导限制性核酸内切酶 (RE) 或用新制癌菌素 (NCS) 治疗，WASP 激活 ARP2/3， 它将核肌动蛋白聚合成分支丝 [4]。这增强了人力资源 DSB 的流动性 以及它们随后聚集到人力资源领域。 DNA 拓扑异构酶 II (Top2) 抑制剂依托泊苷 (ETO) 产生含有蛋白质-DNA 加合物的 DSB， 需要切除并随后通过 HR 因子进行修复，包括 MRN、CtIP 和 BRCA1 [5, 6]。因为 在修复之前绝对需要去除有毒的 Top2，ETO 是探测功能的独特方法 切除与运动的关系。 ETO 用于治疗多种癌症，包括白血病 和软组织癌。然而，治疗与易位引起的继发性白血病有关。使用 通过活细胞成像，我展示了 ETO DSB 经历 ARP2/3 介导的运动和聚集。然而，与 RE 和 NCS DSB，移动不仅限于 G2，也发生在 G1。此外，ETO 在 G1 中破局 进行切除并加载 HR 机械，例如 RPA。我也开始研究人力资源因素的作用， 包括 Mre11 和 BRCA2，在 RE、NCS 和 ETO 生成 DSB 后形成修复域。 尽管 DSB 聚类对于 HR 至关重要，但人们对修复域的形成方式及其局部性知之甚少。 和全基因组的影响。例如，我们并不完全理解运动之间的串扰（肌动蛋白、 WASP）和哺乳动物细胞中的修复（HR 机制）。此外，DSB 的动态可能会影响 染色体重排。我们的实验室正在整合高通量基因组技术来评估基因 基因相互作用和易位事件，以确定 DSB 迁移性的全基因组影响。这 本研究的总体目标是阐明核肌动蛋白聚合和 HR 的机制 蛋白质调节修复域的形成并评估 DSB 迁移性对全基因组的影响。我 假设 HR 蛋白（包括切除机制）在调节 ARP2/3 中发挥关键作用- 介导的 DSB 运动和随后的聚类。我进一步提出核肌动蛋白聚合 影响 DNA 损伤后的基因组组织，从而影响易位频率。我会调查 这些假设的目的如下： 目标 1：阐明 HR 机制对 Arp2/3 依赖的 DSB 聚类的贡献。 目标 2：确定 ARP2/3 介导的 DSB 运动对基因组稳定性的影响。