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.

项目摘要：DNA双链断裂（DSB）修复在空间上组织到核修复域中 这特别促进了通过同源重组（HR）的DSB修复。人力资源，主要DSB途径之一 与非同源末端结合在一起，还与肿瘤发生有关，特别是遵循突变 肿瘤抑制基因BRCA1和BRCA2 [1，2]。我们的实验室证明，在DSB形成时 诱导限制性核酸内切酶（RE）或用新核酸盐（NCS）处理，WASP激活ARP2/3， 将核肌动蛋白聚合到分支细丝中[4]。这增强了原定为人力资源的DSB的移动性 然后它们随后聚集到人力资源域。 DNA拓扑异构酶II（TOP2）抑制剂依托泊苷（ETO）产生携带蛋白DNA加合物的DSB 需要通过人力资源因素进行切除和随后的修复，包括MRN，CTIP和BRCA1 [5，6]。因为 在维修之前，绝对需要中毒的TOP2去除，ETO是探测功能的独特方法 切除与运动之间的关系。 ETO用于治疗广泛的癌症，包括白血病 和软组织癌。但是，由于易位，治疗与继发性白血病有关。使用 实时成像，我表明ETO DSB经历了ARP2/3介导的运动和聚类。但是，与众不同 RE和NCS DSB，运动不限于G2，而是在G1中发生。此外，ETO在G1中断裂 进行切除和负载HR机械，例如RPA。我还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之后的修复域形成中。 尽管DSB聚类对于HR至关重要，但对如何形成维修域及其本地知识知之甚少 和全基因组的影响。例如，我们不完全理解运动之间的串扰（actin， 哺乳动物细胞中的黄蜂）和修复（HR机械）。此外，DSB的动力可能会影响 染色体重排。我们的实验室正在整合评估基因的高通量基因组技术 基因相互作用和易位事件，以确定DSB迁移率的全基因组意义。这 这项研究的总体目标是阐明核肌动蛋白聚合和人力资源的机制 蛋白质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（包括切除机械）在调节ARP2/3-- 介导的DSB运动和随后的聚类。我进一步建议核肌动蛋白聚合 影响DNA损伤后的基因组组织，从而影响易位频率。我会调查 这些假设在以下目的中： 目标1：阐明人力资源机械对ARP2/3依赖性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。