This is a timely and valuable resource paper that systematically maps phosphorylation dynamics across a broad panel of DNA damage-inducing agents. The study’s main strength is its comparative design: by profiling eleven genotoxic or stress-inducing treatments under matched acute conditions, the authors move beyond a generic “DDR phosphoproteome” and begin to separate phosphorylation programs associated with double-strand breaks, replication stress, and more pleiotropic cellular stress responses. This is particularly useful for the DDR field, where ATM/ATR-dependent phosphorylation is often discussed as a unified response despite clear biological differences between lesion types and treatment contexts.

Several aspects of the work are especially compelling. First, the recovery of known DDR phosphorylation events, including sites on BRCA1, MRE11, CtIP, and EXO1, supports the quality of the phosphoproteomic dataset. Second, the integration of kinase-substrate enrichment, motif analysis, conservation scoring, GO enrichment, and clustering gives the paper strong analytical depth. The observation that many regulated S/T-Q sites remain functionally uncharacterized, including sites on RNA-processing, chromatin-associated, and proteostasis-related proteins, is important and should stimulate follow-up work. Third, the phospho-cluster and IDR analysis adds a mechanistic layer that is often missing from large-scale phosphoproteomic studies. The idea that DDR-regulated phosphorylation clusters may alter local charge distribution, intramolecular contacts, and potentially condensate behavior is interesting and biologically plausible. Finally, the focused validation of UBE3A S218 phosphorylation by ATM/ATR and its effect on γH2AX signaling and genotoxic stress survival provides a strong example of how the resource can nominate functionally relevant DDR regulators.

There are several points that would strengthen the manuscript further. The study relies heavily on U2OS cells, which are widely used in DDR research but have cancer-specific genetic and signaling features. The authors should be more explicit about which conclusions are likely to be generalizable versus cell-line specific. Validation of selected phosphorylation signatures, especially the UBE3A findings or representative RNA-processing/proteostasis targets, in an additional cell model would increase confidence. Relatedly, because several treatments have pleiotropic effects beyond DNA damage, the authors should more clearly distinguish “DNA damage response” from broader stress-response phosphorylation throughout the manuscript. The FA, MMS, H₂O₂, and AsO₂ results are interesting precisely because they reveal ribotoxic/MAPK-associated signaling, but the terminology should avoid implying that all observed phosphorylation changes are directly DNA damage-driven.

The UBE3A section is one of the most mechanistically developed parts of the paper, but the model would benefit from more direct evidence connecting S218 phosphorylation to UBE3A activity, localization, proteasome association, or substrate ubiquitylation. The survival and γH2AX rescue data are convincing as functional readouts, but they do not yet define the molecular mechanism. Similarly, the phospho-cluster/IDR simulations are intriguing, but the paper should be careful not to overstate functional consequences without experimental validation of conformational or interaction changes. These analyses are best presented as strong hypotheses generated from the phosphoproteomic resource.

Overall, this manuscript provides a rich and well-executed phosphoproteomic atlas of the acute cellular response to diverse genotoxic agents. Its major contribution is not only the identification of regulated phosphorylation sites, but the organization of these sites into lesion-associated, pleiotropic, kinase-linked, and biophysically meaningful categories. With clearer framing around treatment-specific versus canonical DDR responses, and with some additional validation or discussion of cell-context limitations, this work would be a useful resource for researchers studying genome stability, replication stress, RNA-processing in the DDR, and proteostasis regulation after DNA damage.