Homologous Recombination Repair Domains: Formation and Impact on Genome Stability

项目摘要：DNA双链断裂(DSB)修复在空间上被组织成核修复结构域 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 与非同源末端连接一起，与肿瘤的发生有关，特别是在 肿瘤抑制基因BRCA1和BRCA2[1，2]。我们的实验室通过以下方式证明了DSB的形成 诱导限制性内切酶(RE)或用新卡西诺抑素(NCS)处理，WASP激活Arp2/3， 它将核肌动蛋白聚合成分支细丝[4]。这增强了发往HR的DSB的移动性 并随后将其聚集到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， 需要通过HR因素进行切除和后续修复，包括MRN、CtIP和BRCA1[5，6]。因为 修复前绝对需要去除中毒的TOP2，ETO是探测功能性TOP2的唯一方法 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。vbl.使用 活体细胞成像显示，ETO DSB经历了Arp2/3介导的运动和聚集。然而，不同于 RE和NCS DSB，运动不仅限于G2，而且也发生在G1。此外，ETO在G1中中断 接受切除并加载HR机械，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产生DSB后的修复结构域形成中。 尽管DSB集群对人力资源至关重要，但人们对修复域是如何形成的以及它们的局部情况知之甚少 以及全基因组的影响。例如，我们没有完全理解运动(肌动蛋白， 黄蜂)和修复(HR机制)在哺乳动物细胞中。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流动性的全基因组含义。这个 这项研究的主要目标是阐明核肌动蛋白聚合和HR 蛋白质调节修复结构域的形成，并评估DSB移动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- 介导的DSB移动和随后的聚集。我进一步提出，核肌动蛋白聚合 在DNA损伤后影响基因组组织，从而影响易位频率。我会调查的 这些假设的目的如下： 目的1：阐明HR机制在Arp2/3依赖的DSB聚集中的作用。 目的2：确定Arp2/3介导的DSB运动对基因组稳定性的影响。

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.