br Author contributions br Acknowledgments TJC KW and

Author contributions
Acknowledgments TJC, KW, and VG are supported by awards made to MJN: a Consolidator Grant from the European Research Council under Grant no. 311336; a University Research Fellowship from the Royal Society, and a Career Development Award from the Human Frontiers Science Program Organisation.
Introduction DNA replication is a tremendously challenging, time-consuming, and vital task for eukaryotic organisms. The maintenance of genomic integrity during this process is challenged by endogenous and exogenous factors that cause replication forks to slow and stall, and, in extreme cases, this leads to DNA breakage (Halazonetis et al., 2008). Cells are equipped with a complex DNA damage response (DDR) consisting of protein networks that enable them to cope with replication stress (RS), and a malfunction in these systems can result in genomic instability and oncogenesis (Jackson and Bartek, 2009). These protective signaling pathways require the precise spatial and temporal coordination of DDR components, which is achieved by dynamic and specific post-translational modifications (PTMs) (Polo and Jackson, 2011). In particular, protein Phentolamine Mesylate sale is a well-established driver of the RS response, with the ATR (ataxia telangiectasia and Rad3-related protein) kinase functioning as the key initiator and orchestrator (López-Contreras and Fernandez-Capetillo, 2010, Shiloh, 2001). Depletion of this central kinase leads to replication fork breakage and genomic instability, instigating a phosphorylation response mounted by the ATM (ataxia telangiectasia mutated) kinase, which mediates repair and checkpoint activation upon double-strand breaks (DSBs) (Murga et al., 2009, Smith et al., 2010). ATM and ATR belong to the same atypical serine/threonine kinase family (the phosphatidyl inositol 3′ kinase-related kinases [PIKK]-related kinases) with similar substrate sequence specificity (Kim et al., 2009), but they have unique triggers. Although ATR responds to the accumulation of single-stranded DNA (ssDNA) and regulates replication, ATM is the key mediator of the cellular response to DSBs. DNA-dependent protein kinase (DNA-PK) is the third member of this kinase family; however, its functions are confined to local repair processes (Meek et al., 2008). Phosphorylation, however, must act in concert with other PTMs, such as ubiquitylation, to elicit efficient responses to genotoxic insults (Ulrich and Walden, 2010). The functions of PTMs in the DNA damage and RS responses have therefore been subject of intense investigations, individually (Beli et al., 2012, Bennetzen et al., 2009, Danielsen et al., 2011, Jungmichel et al., 2013) and in concert (Gibbs-Seymour et al., 2015, González-Prieto et al., 2015, Hunter, 2007). More recently, studies have revealed the significance of protein SUMOylation in the DDR, and deregulation of the small ubiquitin like modifier (SUMO) system has been shown to confer genomic instability (Bergink and Jentsch, 2009, Bursomanno et al., 2015, Jackson and Durocher, 2013, Xiao et al., 2015). Using various RS-inducing agents, these studies have shown that the SUMOylation status of a number of proteins is modulated when DNA replication is perturbed (García-Rodríguez et al., 2016). Furthermore, it has been demonstrated that phosphorylation and SUMOylation intersect at various levels (Gareau and Lima, 2010). A phosphorylation-dependent SUMO modification (PDSM) motif has been suggested to prime SUMOylation (Hietakangas et al., 2006) by enhancing the binding of the SUMO E2 enzyme UBC9 (Mohideen et al., 2009), and phosphorylation was also found to regulate the function of SUMO-interacting motifs (SIMs) (Stehmeier and Muller, 2009). However, a potential global coordination of the SUMOylation response and the well-known phosphorylation response to RS remains unexplored. Quantitative mass spectrometry (MS)-based proteomics and developments in enrichment methodologies have seen tremendous developments in recent years (Hendriks and Vertegaal, 2016). State-of-the-art MS technologies allow the identification of thousands of SUMOylation sites (Hendriks et al., 2017, Lamoliatte et al., 2014, Lamoliatte et al., 2017, Schimmel et al., 2014, Tammsalu et al., 2014) and tens of thousands of phosphorylation sites from cellular systems (Francavilla et al., 2017, Mertins et al., 2016, Olsen et al., 2010). In this study, we utilized complementary proteomics strategies to identify the interplay between the global SUMOylation and phosphorylation responses to replication stressors. We identified regulation of thousands of phosphorylation sites and hundreds of SUMOylation sites in response to treatment with the DNA inter-strand crosslinking (ICL) agent mitomycin C (MMC) and hydroxyurea (HU), with a number of proteins co-regulated by both PTMs. Our investigations revealed that the well-established apical responders to RS and RS-induced DSBs, ATR and ATM, both modulate protein SUMOylation at various stages of the RS response. Our findings not only identify an intersection between phosphorylation and SUMOylation in the RS response but also reveal further levels of signaling regulation in this response by the two most prominent kinases of the DNA damage and RS responses.