Defining synthetic lethal relationships with loss of the homologous recombination factor Rad52

总结。该项目的长期目标是定义合成致死因子和途径 人类RAD52蛋白的丢失。Rad52在几个同源驱动的DNA修复途径中发挥着重要作用， 包括单链退火、转录偶联同源重组和有丝分裂DNA合成 (迈达斯)。虽然RAD52不是必需的，但RAD52的损失与乳腺癌1(BRCA1)或 乳腺癌2(BRCA2)基因是人工合成的致命性基因。因此，Rad52是一个耐人寻味的潜在治疗靶点。 BRCA缺陷型癌症。然而，创造RAD52状态的所有途径和因素- 当受到损害时的依赖是不被理解的，这项提议的长期目标就是解决这个问题 知识上的差距。在初步数据中，我展示了我的CRISPR基因敲除屏幕在Rad52基因敲除(Rad52KO) 细胞与野生型(Rad52WT)比较，以确定与Rad52合成致死的因子(这里定义为丢失 健身)。然后，我进行了二次筛查，确定了三个最热门的导致持续DNA增加的原因 RAD52KO与RAD52WT：ERCC6L/PICH、DHX9和GLE1的损伤和活力丧失。从这些数据中，我的 总体假设是有丝分裂的关键调节因子(PICH)和RNA代谢因子(DHX9和GLE1)是 由于依赖于Rad52来解决来自不同来源的复制压力，因此与Rad52合成致死。目标 1：确定RAD52和PICH之间的合成致死关系。RAD52通过保护基因组稳定性 在MIDAS中的作用和复制应激的抑制。PICH介导后期超细桥的拆分 (UFBs)，一种单独的减轻复制压力的途径。目标1a：我假设这两条路径是部分 在防止与复制应激有关的遗传毒性损伤积累方面是多余的。也就是说，我假设皮奇- 在RAD52KO细胞中，UFBs将升高，相反，PICH的耗竭将导致RAD52的升高 在有丝分裂细胞以及MIDAS中募集到复制压力。目标1b：我还假设复制强调 持续到有丝分裂成为缺乏RAD52和PICH的细胞中持续DNA损伤的来源。我将通过以下方式进行测试 检测有丝分裂细胞中磷酸化RPA2(PrPA)、FANCD2HAX和γ的定位。目标2：定义 RAD52与RNA代谢因子DHX9和GLE1的合成致死关系DHX9和GLE1 已被证明可以抑制RNA-DNA杂交体(R-环)。因此，我假设与R环相关的复制 压力是这些基因和Rad52之间合成致命性的基础。我会化验这些物质是否会耗尽 基因增加R-环，导致有丝分裂复制应激水平升高(即Rad52积聚到病灶， MIDAS、PICH-UFBs和目标1b中描述的复制应激措施)。总而言之，这些研究将 深入了解这些基因的丢失如何产生对Rad52介导的复制抑制的依赖 在有丝分裂(即MIDAS)中的应激，加深了我们对基因组维持机制的理解，具有长期的- 识别RAD52抑制剂的肿瘤特异性易损性的学期目标。

SUMMARY. The long-term goal of this project is to define factors and pathways that are synthetic lethal with loss of the human Rad52 protein. Rad52 plays essential roles in several homology-driven DNA repair pathways, including single strand annealing, transcription-coupled homologous recombination, and mitotic DNA synthesis (MiDAS). Although Rad52 is not essential, Rad52 loss with disruption of either the breast cancer 1 (BRCA1) or breast cancer 2 (BRCA2) genes is synthetic lethal. Thus, Rad52 is an intriguing potential target for treatment of BRCA-deficient cancers. However, the full breadth of pathways and factors that create a state of Rad52- dependence when compromised are not understood, and the long-term goal of this proposal is to address this gap in knowledge. In preliminary data, I present my CRISPR knock-out screen in Rad52 Knock-out (Rad52KO) cells vs. wild-type (Rad52WT) to identify factors that are synthetic lethal with Rad52 (defined here as loss of fitness). I then present secondary screening that identified three top hits causing increased persistent DNA damage and loss of viability in the Rad52KO vs Rad52WT: ERCC6L/PICH, DHX9 and GLE1. From these data, my overall hypothesis is that a key regulator of Mitosis (PICH) and RNA metabolism factors (DHX9 and GLE1) are synthetic lethal with Rad52 due to dependence on Rad52 to resolve replication stress from diverse sources. Aim 1: To define the synthetic lethal relationship between Rad52 and PICH. Rad52 protects genome stability through roles in MiDAS and suppression of replication stress. PICH mediates resolution of anaphase ultrafine bridges (UFBs), a separate pathway to mitigate replication stress. Aim 1a: I posit that these two pathways are partially redundant in preventing accumulation of genotoxic damage tied to replication stress. Namely, I posit that PICH- UFBs will be elevated in Rad52KO cells, and conversely that depletion of PICH will cause elevated Rad52 recruitment to replication stress in mitotic cells, as well as MiDAS. Aim 1b: I also posit that replication stress that persists until mitosis is the source of persistent DNA damage in cells lacking Rad52 and PICH. I will test this by assaying phosphorylated RPA2 (pRPA), γH2AX, and FANCD2 localization in mitotic cells. Aim 2: To define the synthetic lethal relationship between Rad52 and RNA metabolism factors DHX9 and GLE1. DHX9 and GLE1 have been shown to suppress RNA-DNA hybrids (R-loops). Thus, I hypothesize that R-loop-related replication stress underlies synthetic lethality between these genes and Rad52. I will assay whether depletion of these genes increases R-loops, causes elevated levels of mitotic replication stress (i.e. Rad52 accumulation into foci, MiDAS, PICH-UFBs, and the measures of replication stress described in Aim 1b). In summary, these studies will provide insight into how loss of these genes create a dependence on Rad52-mediated mitigation of replication stress in mitosis (i.e., MiDAS), enhance our understanding of genome maintenance mechanisms, with a long- term goal of identifing tumor-specific vulnerabilities for Rad52 inhibitors.