Dissecting the DNA Damage Response with Functional Proteomics - SUMMARY The DNA damage response (DDR) entails a tightly choreographed reorganization of protein complexes involved in DNA repair and damage checkpoints. Post-translational modifications (PTMs) fulfil a crucial role in the DDR. However, the mechanisms by which these changes in proteome are dynamically controlled remain elusive. Particularly, much remains to be learned regarding which kinases other than well-characterized ones (e.g., ATM, ATR, DNA-PKcs) are involved in the process, how protein phosphorylation is erased by phosphatases, and how DDR proteins temporally regulate the recruitment and activity of kinases and phosphatases are not clearly understood. We have developed state-of-the-art mass spectrometry (MS)-based functional proteomics tools to globally monitor and quantify changes in protein abundance, protein-protein interactions (PPIs), and PTMs within the context of DDR, facilitating identification of novel regulators and mediators of DDR and providing mechanistic insights into their functions. With quantitative proteomics and systems biology tools combined with biochemical and cell biological approaches established in my laboratory, this MIRA R35 research program aims to define the functional interactome of DDR proteins and mechanistically delineate PTM circuitries to better understand DNA repair mechanisms. Specifically, we will investigate the mechanism by which the ARAF kinase regulates DDR through interaction with RAD51D and examine DNA repair defects associated with ARAF depletion and/or kinase activity inhibition to define a novel DDR-regulatory kinase pathway. Moreover, based on our own published results (Kim et al, Science, 2021, PMID:34591612) implicating Spinophilin (SPN), a substrate targeting subunit of protein phosphatase PP1, in the DRR via its association with the tumor suppressor BRCA1-BARD1, we will analyze the spectrum and kinetics of SPN-PP1-mediated protein dephosphorylation within DDR by analyzing the phospho- proteomes of SPN wild-type and mutants cells. Lastly, based on our preliminary interactome data that SPN interacts with RSBN1L (histone H4K20me2 demethylase), we will investigate whether BRCA1 recruits RSBN1L through its interaction with SPN to DNA double strand break sites to demethylate H4K20me2, which we surmise to create a chromatin environment conducive for BRCA1-BARD1 recruitment. We will also analyze whether RSBN1L regulates the choice of DSB repair pathways between HR and NHEJ by regulating the chromatin association of BARD1 and 53BP1. Our research program will capture dynamic PPIs and PTMs that help ensure the timely execution of DDR and dissect novel DDR regulatory mechanisms.
