Cells incur DNA damage from both endogenous cellular metabolism and exogenous sources. The highly conserved DNA damage response (DDR) pathway monitors the genomic integrity of the cell and protects against genotoxic stresses. Genome instability is a common characteristic of cancer cells, and components of the DDR machinery are often mutated in cancer. In Saccharomyces cerevisiae, DNA damage activates the sensor kinases, Mec1 and Tel1 (ATR and ATM in human, respectively). The response is further amplified by activation of the effector kinase, Rad53, to regulate a variety of cellular processes, including cell cycle progression, DNA damage repair, chromatin remodeling, and transcription.
To improve our understanding of the global response to DNA damage, we sought to identify targets of Rad53 using a mass spectrometry-based phosphoproteomic screen in a strain deleted for phosphatases known to counteract checkpoint kinases. Of the top 40 hits, we found that 17 display DNA damage-dependent mobility shifts on SDS-PAGE gels and determined that Rad53 phosphorylates 22 out of the 40 hits by in vitro kinase assay. Many of the hits are proteins that are involved in RNA metabolism. In particular, we are following up on Xrn1, a highly conserved 5’ exoribonuclease, which functions primarily in mRNA decay. Xrn1 is modified upon DNA damage in a checkpoint-dependent manner and is directly phosphorylated by Rad53 in vitro. We used SILAC to identify proteins that interact with Xrn1 in a RAD53-dependent manner. Preliminary data suggests that the association of Lsm1 and Lsm3 with Xrn1 is reduced in a rad53∆. In addition, we have found that at least one member of the decapping complex rescues the strong damage sensitivity of an xrn1∆. We are currently conducting an unbiased screen to identify other suppressors of this mutant to determine the pathway relevant to its damage sensitivity. We hypothesize that the DDR regulates mRNA turnover by targeting Xrn1 in response to damage.