Prediction and Investigation of Novel Proteins in DNA Double Stranded Break Repair

Abstract

DNA double stranded breaks (DSBs) are the most genotoxic forms of DNA lesions, causing fragmentation of the DNA strands. Mis-repaired and unrepaired DSBs lead to chromosomal rearrangement and genomic instability promoting tumorigenesis or cell death. DSBs are primarily repaired by two independent and highly conserved pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). HR requires a homologous sequence to repair DNA breaks, whereas NHEJ repair is achieved through direct ligation of the broken ends of DNA. The process of NHEJ involves three main protein complexes: Yku70/Yku80 initiates and stabilizes the DNA ends, Mre11/Rad50/Xrs2 brings broken ends to close proximity and Dnl4/Lif1/Nej1 ligates the DNA ends. Protein-protein interaction (PPI) has been utilized in functional genomics studies to identify novel proteins involved in different pathways based on their PPI profiles. In this study we aim to screen for novel proteins involved in NHEJ using PPI predictions. We use a computational tool to predict novel PPIs between DNA repair proteins and human proteome. Using this method, we predicted 271 novel PPIs, expanding the reported human DNA repair interactome by 75%. Yeast homologs of the novel human gene candidates were subjected to a plasmid-based repair assay, in which deletion of eight of 12 yeast genes showed reduction in the repair efficiency. We further investigated the roles of Tpk1, Arp6 and Psk1/Psk2 in NHEJ repair. Deletion of any one of TPK1, ARP6, PSK1 and PSK2 reduced efficiency of repair in chromosomal and plasmid based assays and showed sensitivity to different DNA damaging agents. We suggest that Tpk1, catalytic subunit of protein kinase A, is involved in recruitment of Nej1 to the site of damage and its role in NHEJ is dependent on Nej1. We propose that the involvement of actin related protein 6, Arp6, in NHEJ is linked to Rsc2 and iii Mre11. Additionally, we suggest a role for PAS kinase complex, Psk1/Psk2, that is connected to DNA damage checkpoint proteins. First and foremost, I would like to thank Dr. Golshani for giving me the opportunity to work in his lab. I would like to express my gratitude for his support, guidance and mentorship. But mostly thanks for your patience and believing in me, and for helping me to reach goals that would've not been possible without your supervision, inspiration and encouragement.