High throughput screening approach for identifying Proteins Whose Proteasomal Degradation is Essential for Double-Strand Break Repair

DNA double strand breaks (DSBs) are dangerous lesions that, if improperly repaired, may lead to chromosomal rearrangements and other genetic alterations, and eventually to cancer. Previous studies have suggested a role for the Ubiquitin-Proteasome System (UPS) in DSB repair and have speculated that its proteolytic activity may be required to degrade proteins of the DSB repair machinery that still await discovery. In this study we devised a systematic robotic screening approach for identifying yeast and human proteins involved in DSB repair that are regulated via their proteasome-mediated degradation. We use an arrayed collection of more than 5000 yeast strains, each over-expressing (OE) a different yeast ORF when grown on galactose as a carbon source. Our screening methodology is based on the following rationale: (i.) If the turnover of a specific protein (defined here as A) is mediated by the proteasome, then its specific OE may mimic the situation in which a defective/inactive proteasome leads to accumulation of A in the nucleus. (ii.) If the OE of A impairs cell growth when combined with DSBs, induced by the CRISPR\Cas9 system, it may suggest A as a candidate protein that should be degraded by the UPS to allow proper DSB repair. We expect that such a list will facilitate the understanding of the regulated removal of repair proteins from DNA, a broad problem that currently is not well understood. It will also serve as the basis for detailed mechanistic follow-up studies as well as for novel therapeutic approaches.