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

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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.**