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: 10212281
• 负责人: Jennifer Ashley Zagelbaum
• 金额: $ 4.6万
• 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/10212281
• 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/antagonist; 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.

DNA双链断裂（DSB）修复在空间上被组织成核修复结构域 其通过同源重组（HR）特异性地促进DSB修复。HR，DSB的主要途径之一 沿着非同源末端连接，与肿瘤发生有关，特别是在 肿瘤抑制基因BRCA 1和BRCA 2 [1，2]。我们的实验室证明，在DSB形成时， 限制性内切酶（RE）诱导或用新抑癌素（NCS）处理，WASP激活ARP 2/3， 其将核肌动蛋白聚合成分支丝[4]。这提高了指定用于人力资源的DSB的流动性 以及它们随后聚类到HR域中。 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， 需要切除和随后的HR因素修复，包括MRN、CtIP和BRCA 1 [5，6]。因为 绝对要求在修复前清除中毒的Top2，ETO是探测功能的独特方法 切除和移动之间的关系。ETO用于治疗多种癌症，包括白血病 和软组织癌。然而，治疗与由于易位引起的继发性白血病有关。使用 活细胞成像，我表明，ETO DSB进行ARP 2/3介导的运动和集群。但不同于 RE和NCS DSB，移动不限于G2，也发生在G1。此外，G1中ETO断裂 进行切除并装载HR机械，例如RPA。我也开始研究人力资源因素的作用， 包括Mre 11和BRCA 2，在RE、NCS和ETO产生DSB后修复结构域形成中的作用。 尽管DSB聚类对于HR至关重要，但对修复结构域如何形成及其局部分布知之甚少。 和全基因组的影响。例如，我们不完全理解运动（肌动蛋白， WASP）和修复（HR机制）。此外，争端解决机构的动态可能会影响 染色体重排我们的实验室正在整合高通量基因组技术，评估基因- 基因相互作用和易位事件，以确定全基因组的影响DSB的流动性。的 本研究的首要目标是阐明核肌动蛋白聚合和HR 蛋白调节修复结构域的形成，并评估DSB迁移率的全基因组影响。我 假设HR蛋白，包括切除机制，在调节ARP 2/3中起关键作用- 介导的DSB运动和随后的聚类。我进一步提出， 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： 目的1：阐明HR机制对Arp 2/3依赖性DSB聚类的贡献。 目的2：确定ARP 2/3介导的DSB运动对基因组稳定性的影响。