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

生物体的DNA总是受到外部力量的攻击，例如暴露在化学品和辐射中。在这种接触可能导致的许多有害事件中，DNA双链断裂(DSB)的形成是最严重的，因为它会导致染色体末端断裂，从而触发染色体融合和重排、细胞周期停滞和细胞凋亡，具体取决于细胞类型和细胞环境。存在两条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(HR)的途径和基于典型非同源末端连接(CNHEJ)的途径。在DSB修复过程中，HR介导的事件需要一个同源序列作为模板，从而导致高保真修复，从而保持遗传信息的准确性。然而，并不是所有的细胞周期都有同源的修复模板，当未复制的染色体没有姐妹染色单体作为修复模板时，cNHEJ在G1期的DSB修复中起着关键作用。在NHEJ期间，断裂的染色体末端被Ku70/80的核心复合体连接在一起，处理后进行连接(这必然会导致可能发生突变的遗传信息的丢失)，并由LIG4连接。这两条DNA修复途径都需要额外的因子参与，然而，HR辅助因子在不同的生物中显示出高度的保守性，这在NHEJ途径中没有观察到。 虽然脊椎动物需要几个NHEJ辅助因子，但线虫一直被认为只使用Ku/LIG4核心复合体进行NHEJ介导的DSB修复。在这个应用中，我们描述了我们在野生型N2var中发现的突变分离。布里斯托尔种群，当线虫在幼虫早期阶段受到辐射时，会导致生育能力和NHEJ途径中典型的突变体的表型丧失。H19N07.3编码一种仅在线虫物种中存在的小蛋白，并为研究物种特异性因子共同作用为高度保守的NHEJ复合体有效修复DSB创造合适的环境提供了独特的机会。 我们建议验证这一假设，并研究H19N07.3及其相互作用蛋白在NHEJ中在体细胞和生殖系组织以及有机体生育中的功能。此外，我们的初步数据表明，H19N07.3的丢失会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，并使我们能够使用线虫系统中可用的强大工具来考虑NHEJ对生育的进化影响。

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.