The regulation and specificity of the XPG-related nucleases in the initiation of the DNA damage response

DNA damage comes in many varieties, but feeds into a limited number of repair and checkpoint pathways. This suggests the formation of a common intermediate as the initiating event in the mounting of a DNA damage response. In this thesis, I study the regulation and specificity of the XPG-related family of 5'-to-3' exo- /endonucleases, which is comprised of Exo1, Rad2/Fen1, and Ast1. I study the recruitment of these three nucleases to site-specific double-strand breaks (DSBs) and their roles in DSB resection at two sites in the genome. I provide evidence for a hierarchy of recruitment of the nucleases to a site in the rDNA, wherein Exo1 is the primary nuclease recruited to the site, Ast1 responds in the absence of Exo1, and Rad2 responds in the absence of the other two. I also show that the nucleases are required for the response to replication stress. Nuclease deletions sensitize strains deficient in fork protection complex members and fork processing enzymes to DNA damaging agents, and are required for the survival and replication restart of cells experiencing fork collapse. In addition, I show a novel role for DNA topoisomerase I in checkpoint activation and replication restart during replication stress.