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

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（这必然导致遗传信息的丢失），并由LIG4连接。虽然已知这两种途径都需要辅助因子，但HR辅助因子在不同生物中表现出一定程度的保守性，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通过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环境，通过高度保守的NHEJ复合物有效修复DSB。为了验证这一假设，我们将研究H19N07.3及其相互作用蛋白在NHEJ中的功能，2)该蛋白和NHEJ在不同组织中的作用，3)该蛋白和NHEJ在跨代生育中的作用。这些分析将使我们能够研究生物在发育过程中如何对DNA损伤做出反应，并允许我们使用秀丽隐杆线虫系统中可用的强大工具来考虑NHEJ对生育能力的进化影响。

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), and ligated by LIG4. While both pathways are known to require accessory factors, the HR accessory factors show a 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 with 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 a context for efficient DSB repair by the highly conserved NHEJ complex. We propose to test this hypothesis and 1) investigate the function of H19N07.3 and its interacting proteins in NHEJ, 2) the role of the protein(s) and NHEJ in different tissues, 3) and the function of the protein(s) and NHEJ in transgenerational fertility. These analyses will enable us to examine how organisms respond to DNA damage in the context of development and allow us to consider the evolutionary impact of NHEJ on fertility using the powerful tools available to us in the C. elegans system.