The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.Cells are highly responsive to their environment, especially DNA damaging agents. Damaged DNA in cells is rapidly sensed and turned into signals by the DNA damage checkpoint to control many processes, including cell cycle progression, DNA replication and repair, and gene transcription (1). The DNA damage checkpoint consists of several evolutionarily conserved protein kinases (2, 3). Understanding the function of the DNA damage checkpoint requires knowledge of their in vivo substrates. Although the regulation of DNA damage checkpoint kinases has been studied extensively, the knowledge of their in vivo substrates is limited. This can be attributed to the lack of suitable technology to detect low abundant phosphorylation in cells. With the use of stable isotope labeling, the advancement of high mass resolution mass spectrometry (MS), 3 and the recent development of analytical and computational tools by many laboratories (4 -9), changes in low abundant and regulatory phosphorylation in cells are increasingly detected. Combined with the use of genetics, in vivo kinase substrates have been identified using a quantitative mass spectrometry approach (10).In the yeast Saccharomyces cerevisiae, Mec1 and Tel1, homologs of the mammalian ATR and ATM kinase, respectively, function at the top of the signal transduction cascade in the DNA damage checkpoint (1-3). Mec1 is primarily responsible for the activation of downstream checkpoint kinases including Rad53 (11,12), whereas Tel1 has a more prominent role in regulating telomere length (13). Interestingly, deletion of both MEC1 and TEL1 leads to a synergistic increase in gross chromosomal rearrangements, indicating their redundant role in genome maintenance (14, 15). Mec1 is recruited to the site of DNA damage via replication protein A (RPA) that coats singlestranded DNA. Tel1 on the other hand is recruited by the Mre11-Rad50-Xrs2 complex, which recognizes DNA doublestranded breaks (DSBs) (16 -19). Importantly, DNA DSBs in cells undergo 5Ј to 3Ј resection to generate 3Ј single-...

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel beta-coronavirus that has recently emerged as the cause of the 2019 coronavirus pandemic (COVID-19). Polymerase chain reaction (PCR) based tests are optimal and recommended for the diagnosis of an acute SARS-CoV-2 infection. Serology tests for viral antibodies provide an important tool to diagnose previous exposure to the virus. Here we evaluate the analytical performance parameters of the Diazyme SARS-CoV-2 IgM/IgG serology assays and describe the kinetics of IgM and IgG seroconversion observed in patients with PCR confirmed COVID-19 who were admitted to our hospital. Methods We validated the performance of the Diazyme assay in 235 subjects to determine specificity. Subsequently, we evaluated the SARS-CoV-2 IgM and IgG seroconversion of 54 PCR confirmed COVID-19 patients and determined sensitivity of the assay at three different timeframes. Result Sensitivity and specificity for detecting seropositivity at ≥ 15 days following a positive SARS-CoV-2 PCR result, was 100.0% and 98.7% when assaying for the panel of IgM and IgG. The median time to seropositivity observed for a reactive IgM and IgG result from the date of a positive PCR was 5 days (IQR: 2.75-9 days) and 4 days (IQR: 2.75-6.75 days), respectively. Conclusions Our data demonstrates that the Diazyme IgM/IgG assays are suited for the purpose of detecting SARS-CoV-2 IgG and IgM in patients with suspected SARS-CoV-2 infections. For the first time, we report longitudinal data showing the evolution of seroconversion for both IgG and IgM in a cohort of acutely ill patients in the United States. We also demonstrate a low false positive rate in patients who were presumed to be disease free.

The kinetochore is the central molecular machine that drives chromosome segregation in all eukaryotes. Genetic studies have suggested that protein sumoylation plays a role in regulating the inner kinetochore; however, the mechanism remains elusive. Here, we show that Saccharomyces cerevisiae Ulp2, an evolutionarily conserved SUMO specific protease, contains a previously uncharacterized kinetochore-targeting motif that recruits Ulp2 to the kinetochore via the Ctf3CENP-I-Mcm16CENP-H-Mcm22CENP-K complex (CMM). Once recruited, Ulp2 selectively targets multiple subunits of the kinetochore, specifically the Constitutive Centromere-Associated Network (CCAN), via its SUMO-interacting motif (SIM). Mutations that impair the kinetochore recruitment of Ulp2 or its binding to SUMO result in an elevated rate of chromosome loss, while mutations that affect both result in a synergistic increase of chromosome loss rate, hyper-sensitivity to DNA replication stress, along with a dramatic accumulation of hyper-sumoylated CCAN. Notably, sumoylation of CCAN occurs at the kinetochore and is perturbed by DNA replication stress. These results indicate that Ulp2 utilizes its dual substrate recognition to prevent hyper-sumoylation of CCAN, ensuring accurate chromosome segregation during cell division.

Background: Phosphorylation of Sae2 by Mec1/Tel1 in S. cerevisiae has been shown, but its function was poorly understood. Results: The conserved threonines of Sae2 have a redundant role in DNA damage response, and their phosphorylation directly interacts with Rad53, Dun1, and Xrs2 via their FHA domains. Conclusion: Phosphorylation of Sae2 regulates its DNA repair function. Significance: This work identifies the associated proteins of phosphorylated Sae2.