Regulation of the nonhomologous end joining pathway during DNA repair in C. elegans

线虫 DNA 修复过程中非同源末端连接途径的调节

• 批准号: RGPIN-2019-06071
• 负责人: Zetka, Monique
• 金额: $ 2.33万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745962
• 关键词: Regulation; nonhomologous; end; joining; pathway

The DNA of organisms is perpetually attacked by exogenous forces such as exposure to chemicals and radiation. Of the many deleterious events that can result from such contact, the formation of DNA double strand breaks (DSBs) is the most serious since it results in broken chromosome ends that can trigger chromosome fusions and rearrangements, cell-cycle arrest, and apoptosis, depending on the cell type and cellular context. Two highly conserved major pathways exist to repair exogenous DNA breaks and can be broadly divided into those based on homologous recombination (HR), and those based on canonical non-homologous end joining (cNHEJ). HR-mediated events require a homologous sequence as a template during DSB repair and consequently result in high-fidelity repair that conserves the accuracy of genetic information. However, a homologous repair template is not available in all cell-cycle contexts, and cNHEJ plays a critical role in DSB repair during G1 phase, when unreplicated chromosomes have no sister chromatid to use as a repair template. During NHEJ, broken chromosome ends are held together by a core complex of Ku70/80, processed for ligation (which necessarily results in loss of genetic information that can be mutagenic), and ligated by LIG4. Both DNA repair pathways require the participation of additional factors, however, the HR accessory factors show a high level of conservation across diverse organisms that is not observed in in the NHEJ pathway. While vertebrates are known to require several NHEJ accessory factors, the nematode C. elegans has been thought to proceed with NHEJ-mediated repair of DSBs using only the Ku/LIG4 core complex. In this application, we describe our discovery of mutation segregating in the wild-type N2 var. Bristol population that confers radiation-induced loss of fertility and phenotypes typical of mutants in the NHEJ pathway when the worms were irradiated at early larval stages. H19N07.3 encodes a small protein found only in Caenorhabditis species and provides a unique opportunity to investigate the hypothesis that species-specific factors co-operate to create an appropriate context for efficient DSB repair by the highly conserved NHEJ complex. We propose to test this hypothesis and investigate the function of H19N07.3 and its interacting proteins in NHEJ in somatic and germline tissues and in organismal fertility. Furthermore, our preliminary data indicate that loss of H19N07.3 results in transgenerational loss of fertility that can be partially rescued by chromatin remodelling and we intend to investigate the basis of this phenomenon. These analyses will enable us to investigate how organisms respond to DNA damage in the context of differentiated cell types and development stage and allow us to consider the evolutionary impact of NHEJ on fertility using the powerful tools available to us in the C. elegans system.

生物体的DNA不断受到外部力量的攻击，比如暴露在化学物质和辐射中。在这种接触可能导致的许多有害事件中，DNA双链断裂（DSBs）的形成是最严重的，因为它会导致染色体末端断裂，从而引发染色体融合和重排，细胞周期阻滞和细胞凋亡，这取决于细胞类型和细胞环境。存在两种高度保守的修复外源DNA断裂的主要途径，大致可分为基于同源重组（homologous recombination， HR）的途径和基于规范非同源末端连接（canonical non-homologous end joining, cNHEJ）的途径。在DSB修复过程中，hr介导的事件需要同源序列作为模板，从而导致高保真修复，从而保持遗传信息的准确性。然而，同源修复模板并不是在所有细胞周期环境中都可用，而在G1期，当未复制的染色体没有姐妹染色单体用作修复模板时，cNHEJ在DSB修复中起着关键作用。在NHEJ过程中，断裂的染色体末端由Ku70/80核心复合体连接在一起，进行结扎处理（这必然导致可能导致突变的遗传信息丢失），并由LIG4连接。两种DNA修复途径都需要其他因子的参与，然而，HR辅助因子在不同生物体中表现出高度的保守性，这在NHEJ途径中没有观察到。虽然已知脊椎动物需要几种NHEJ辅助因子，但人们认为秀丽隐杆线虫仅使用Ku/LIG4核心复合物进行NHEJ介导的dsb修复。在这个应用中，我们描述了我们在野生型N2变种布里斯托尔种群中发现的突变分离，当蠕虫在幼虫早期阶段受到辐射时，会导致NHEJ途径中突变体的典型表型和繁殖能力的丧失。H19N07.3编码一种仅存在于杆状线虫物种中的小蛋白，并提供了一个独特的机会来研究物种特异性因子合作创造一个适当的环境，通过高度保守的NHEJ复合物有效修复DSB。我们拟验证这一假设，并研究H19N07.3及其在NHEJ中的相互作用蛋白在体细胞和种系组织以及有机生殖中的功能。此外，我们的初步数据表明，H19N07.3基因的缺失会导致生育能力的跨代丧失，这可以通过染色质重塑部分修复，我们打算研究这一现象的基础。这些分析将使我们能够研究生物如何在分化细胞类型和发育阶段的背景下对DNA损伤作出反应，并使我们能够利用我们在秀丽隐杆线虫系统中可用的强大工具考虑NHEJ对生育能力的进化影响。