Identification and characterization of novel genes involved in DNA double strand break repair process in the yeast Saccharomyces cerevisiae

DNA Double Strand Break (DSB) is the most severe form of DNA lesion. Unrepaired DSBs and/or mutations in key DSB repair genes can lead to genomic instability, cancer or even cell death. DSBs can be repaired by two independent pathways; Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Both pathways are highly conserved from yeast to human. HR requires a homologous region to repair the damage but in NHEJ, the break ends are ligated directly. In Saccharomyces cerevisiae, the key protein complexes of the NHEJ pathway are: Yku70p/Yku80p (YKU), which binds to DNA and initiates the process and stabilizes the broken ends, Mre11p/Rad50p/Xrs2p (MRX), which brings broken ends close together and Dnl4p/Lif1p/Nej1p, which have ligase activities.In this thesis, we aimed to identify novel proteins that influence the NHEJ process in yeast. To this end we performed a combination of high-throughput as well as low-throughput assays, and followed-up by verifying some of the findings. The initial large-scale plasmid repair screening that we performed revealed approximately 60 novel genes that might involve in the NHEJ process. Among these genes, PPH3/PSY2, HUR1, BUB1/BUB2, and SBP1 were selected for follow up experiments. Small-scale plasmid repair and chromosomal break repair assays were used to verify the involvement of these selected candidates in the efficiency of NHEJ. Deletion of any one of the above selected genes showed a reduction in efficiency of both plasmid repair and chromosomal repair assays. Deletion of those genes also showed increased sensitivity to DNA damaging agents Hydroxyurea (HU) and bleomycin. Our follow-up genetic studies suggested a role for PPH3/PSY2 phosphatase complex in the NHEJ process through interaction with checkpoint kinases Rad53 and Chk1. We also propose a novel role for yeast uncharacterized ORF HUR1, in NHEJ pathway through its association with Nej1. We also report that BUB1/BUB2 may affect the efficiency of NHEJ by limiting cell cycle progression. We suggest their joint participation with Apc9p (APC), Clb2p (cyclin), and Swi4p (SBF) proteins in the NHEJ repair process. Additionally, we hypothesize a role for the translational repressor SBP1 in NHEJ pathway, connecting translation control to NHEJ.