br Results br Discussion Context specific and dynamic post

Results
Discussion Context-specific and dynamic post-translational protein modifications are well-established regulators of the signaling pathways that protect eukaryotic DNA integrity during the tremendous task of replication. Advancements in speed, resolution, and sensitivity of MS-based technologies have revolutionized the study of global PTM biology (Olsen and Mann, 2013). With this rise in global PTM data, it has become evident that efficient cellular responses, such as those that safeguard genomic integrity, require the precise and timely coordination of several PTMs and the different nm to lb that regulate them (Papouli et al., 2005). Integrated analysis of PTMs is therefore pertinent for our understanding of the molecular mechanisms that respond to DNA damage and RS. Using state-of-the-art proteomics methodologies, we mapped nearly 1,400 regulated SUMOylation acceptor sites and 3,300 regulated phosphorylation sites in response to the chemotherapeutic agents MMC and HU. Our study reveals that SUMOylation is regulated by the most dominant, apical DDR kinases ATR and ATM, which are known to initiate and coordinate the phosphorylation responses to RS and replication fork breakage. In accordance with previous studies, we found that RS elicits increased SUMOylation of the core ATR-activating proteins, including TOPBP1 and ATRIP. Interestingly, previous studies have shown that the SUMOylation of ATR and its constitutive interactor ATRIP is necessary for efficient ATR-dependent checkpoint signaling (Wu and Zou, 2016, Wu et al., 2014). Further, here we showed that TOPBP1, a key co-activator of ATR, undergoes increased SUMOylation in response to MMC-induced RS. This indicates that SUMOylation of this factor, in addition to that of ATR and ATRIP, may be important for ATR-dependent checkpoint signaling. However, further biochemical and molecular biological analyses are required to confirm the precise role of TOPBP1 SUMOylation in ATR activation. In addition, our data suggest that SUMOylation is a common and relevant modification of a number of proteins involved in ATR activation in response to RS. We aimed to uncover the interplay between phosphorylation and SUMOylation of protein networks in the RS response. Using an integrated proteomics approach, we found that protein SUMOylation was widely modulated by the main regulatory kinases that mediate the phosphorylation response. Parallel proteomics analysis of changes in these two PTMs revealed co-regulation of a number of central RS and DDR responders, including BRCA1, BARD1, and TOPBP1. BRCA1 SUMOylation and phosphorylation have individually been found to play a key role in the function of this protein because SUMOylation has been shown to increases its ubiquitin ligase activity (Morris et al., 2009). It will be interesting in future analyses to determine whether there is co-dependency or cross-regulation of these modifications in the proteins that harbor both phosphorylation and SUMOylation and, in particular, the relevance of this for the functions of central DDR proteins. Our approach, to study co-regulated SUMO- and phospho-modified proteins, proved to be successful to identify key co-modified targets. TOPBP1 was the most highly co-regulated protein in our dataset upon 8 hr of MMC treatment, and we found that TOPBP1 SUMOylation was heavily modulated by ATR inhibition during RS and by ATM upon replication fork breakage. We found this particularly interesting because these central DDR kinases (particularly ATM) are well known to orchestrate various PTM-based networks upon threats to the DNA (Smith et al., 2010). However, the effect of the apical DDR kinases on global protein SUMOylation in response to DNA damage and replication stress has not yet been shown. We determined that such regulation by kinases not only applies to TOPBP1 but, further, to over 800 nearly 1,400 SUMOylation acceptor sites in response to HU-induced RS, demonstrating global regulation of SUMOylation by these kinases in the maintenance of genome stability. Interestingly, we observed decreased SUMOylation of a large subset of proteins upon ATM inhibition under conditions that induce replication fork breakage. This suggests that ATM may be important for global deSUMOylation to maintain and control physiological levels of protein SUMOylation.