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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684414
• 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，例如暴露于化学品和辐射。在这种接触可能导致的许多有害事件中，DNA双链断裂（DSB）的形成是最严重的，因为它导致断裂的染色体末端，这可以触发染色体融合和重排、细胞周期停滞和凋亡，这取决于细胞类型和细胞环境。存在两种高度保守的主要途径来修复外源性DNA断裂，并且可以大致分为基于同源重组（HR）的途径和基于典型非同源末端连接（cNHEJ）的途径。HR介导的事件在DSB修复过程中需要同源序列作为模板，因此导致高保真修复，从而保持遗传信息的准确性。然而，同源修复模板并不适用于所有细胞周期背景，并且cNHEJ在G1期期间的DSB修复中起关键作用，此时未复制的染色体没有姐妹染色单体用作修复模板。在NHEJ过程中，断裂的染色体末端通过Ku 70/80的核心复合物保持在一起，进行连接处理（这必然导致可能具有诱变性的遗传信息的丢失），并通过LIG 4连接。这两种DNA修复途径都需要额外因子的参与，然而，HR辅助因子在不同生物体中显示出高水平的保守性，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，但线虫C.线虫被认为仅使用Ku/LIG 4核心复合物进行NHEJ介导的DSB修复。在本申请中，我们描述了我们在野生型N2变种中发现的突变分离。布里斯托种群，当蠕虫在早期幼虫阶段接受辐照时，会导致辐射诱导的生育力丧失和NHEJ途径中突变体的典型表型。H19N07.3编码一种仅在小杆线虫物种中发现的小蛋白，并提供了一个独特的机会来研究物种特异性因子合作为高度保守的NHEJ复合物的有效DSB修复创造适当背景的假设。我们建议测试这一假设，并调查的功能H19N07.3和它的相互作用蛋白在NHEJ在体细胞和生殖系组织和生物体生育力。此外，我们的初步数据表明，H19N07.3的丢失导致跨代生育能力的丧失，这可以通过染色质重塑来部分挽救，我们打算研究这种现象的基础。这些分析将使我们能够研究生物体如何在分化的细胞类型和发育阶段的背景下对DNA损伤做出反应，并使我们能够使用C中可用的强大工具来考虑NHEJ对生育力的进化影响。elegans系统。**