Probing the spatial organization of DNA damage signaling.

Biochemistry, Molecular & Cell Biology Field Seminar: Mateusz Wagner, Smolka Lab.

Maintenance of genomic stability relies heavily on signaling mediated by the PIKK family kinases ATR, ATM, and DNA-PK. These apical kinases coordinate pathways that control cell cycle checkpoints, replication fork stability, chromatin remodeling, and repair of DNA lesions. Little is understood about how DNA damage signaling events are spatially organized within the nucleus, despite extensive evidence that the nucleus is a highly compartmentalized organelle. For example, it is currently unclear how PIKK activation and signal propagation differ in response to DNA lesions at distinct compartments such as nucleolus, centromeres and telomeres.

Mass spectrometry-based phosphoproteomics is a powerful approach to investigate kinase signaling networks. However, it is currently unable to monitor kinase signaling events with spatial resolution. In addition, typical phosphoproteomic pipelines suffer from incomplete coverage due to the low abundance of many functionally relevant signaling events. Here we developed ProxiPhos, an approach that couples proximity labeling (TurboID), IMAC-based phosphoenrichment, and quantitative mass spectrometry, for multiplexed and spatially-resolved analysis of phosphorylation events.

We applied ProxiPhos for the in-depth analysis of the nuclear phosphoproteome under drug conditions that induce DNA replication stress and activate the DNA damage sensor kinases ATR, ATM and DNA-PKcs. By fusing TurboID to the core replisome component PCNA, we showed that ProxiPhos can detect and monitor a complex and dynamic set of phosphorylation events associated with the DNA replication machinery and perturbed by chemotherapy compounds.

Committee: Marcus Smolka, Brooks Crickard, Aleksandra Skirycz.