SILAC labeling and mass spectral analysis reveals temporal changes in Nbn protein interactions and PTMs in response to DNA damage

摘要

Tcof1 was discovered as a novel binding partner of the MRN complex (Mre11/Rad50/Nbn). Little is known about Tcof1 function in the cellular response to DNA damage but Atm/Atr-mediated phosphorylations have been previously reported in other high-throughput phosphoproteomic studies. Mutations within the TCOF gene in humans is responsible for Treacher Collins-Franceschetti Syndrome, a disease that shares craniofacial developmental defects with the Nbn mutants related to Nijmegen Breakage Syndrome. SILAC ratios of Mre11 and Rad50 coincide at each time point analyzed suggesting that Mre11 and Rad50 form a complex before they associate with Nbn or, alternatively the three components of the MRN complex come together to form the complex simultaneously. Tcof1 associates with the MRN complex independent of the MRN complex formation characteristics defined by temporal SILAC ratios of Mre11 and Rad50 and was also independent of IR exposure in NBS-ILB1 cells. Western blot and MS data suggest that only a minor fraction of the MRN complex is assocated with Tcof1 in either irradiated and mock irradiated samples. Methionine cleavage and N-terminal acetylation of S2 on Mre11 is a novel co-translational modification found in this study and has unknown biological significance. Interestingly, Atm-mediated phosphorylation of Nbn S343 and S397 of S phase samples contain the highest levels of phosphorylation among Mock irradiated samples for their respective residues. While Nbn S432 has previously been identified in high-throughput MS analyzes, our study reveals that this site is highly phosphorylated in unirradiated samples and changes in response to DNA damage.