A Genome-scale RNA Interference Screen Identifies Novel Regulators of DNA Double-strand Break Repair

All living organisms are continuously challenged by agents in their normal cellular environment that can inflict damage to their genetic material. DNA damage can have negative implications on cellular fitness by disrupting genomic processes such as DNA replication and gene expression. In addition, DNA lesions can lead to gene mutation and gross chromosomal rearrangements, events that are implicated in the development of cancer. DNA double-strand breaks (DSBs) are considered one of the most severe types of DNA damage. To protect genome integrity, organisms have evolved several DSB repair mechanisms. Homologous recombination (HR) is an essential DSB repair pathway that is of critical importance during the S- and G2-phases of the cell cycle. HR plays a central role in promoting genome stability and preventing tumourigenesis. For example, mutations in the genes coding for the HR factors BRCA1 and BRCA2 are responsible for familial breast and ovarian cancer. The limiting step in the HR pathway is the generation of single-stranded DNA (ssDNA) by DNA end resection. In this thesis, I describe the establishment of an immunofluorescence-based assay that monitors end resection by quantitative image-based cytometry (QIBC). Employing this assay, I conducted a plate-based RNA interference (RNAi) screen using a library that targets 18,452 genes. As expected, the top hits in my screen were known regulators of end resection including CtIP and all three subunits of the MRE11-RAD50-NBS1 (MRN) complex. I also outline the validation of one the strongest candidate end resection activators identified in the screen, the zinc finger protein, ZNF335. I demonstrate that ZNF335 promotes DSB repair by HR through the enhancement of DNA end resection. In the final chapter of this thesis, I describe the establishment of a cell microarray-based platform for conducting RNAi screens. The platform circumvents the long liquid-handling robotic procedures and large quantities of reagents that are common for plate-based screens.Ph.D