Identifying proteins with roles in DNA double strand break repair regulated by their proteasome degradation

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. Defects in DSB repair are observed in various cancers and influence the responsiveness of cancer cells to therapeutic regimens. A growing body of evidence suggests a role for the Ubiquitin-Proteasome System (UPS) in the repair of DSBs, and indicate a physical association of the proteasome with the sites of DSBs. These studies also suggest that the proteasomal proteolytic activity may be required to degrade components of the DSB repair machinery, and impaired DSB repair caused by UPS dysfunction may be attributed to the specific accumulation of additional UPS targets that still await. Our research aims to comprehensively identify such substrates. For this purpose, we devised a systematic genetic robotic screening approach to identify yeast proteins that impair cell growth when overexpressed (OE) in the presence of DNA damaging agents. This approach is based on the following rationale: (i.) If the turnover of a specific protein (defined as A) is mediated by the proteasome, then its specific OE may mimic the situation of a defective/inactive proteasome and leads to the accumulation of A in the nucleus. (ii.) If the OE of A impairs cell growth when combined with DSBs, it may implicate A as a candidate that should be degraded by the UPS for proper DSB repair.

This will help understanding the regulated removal of DSB repair proteins from DNA, moreover, the isolation of additional proteasomal targets with roles in DSB repair will be of great importance to exploiting this therapeutic avenue.