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.

Background. Currently it is unknown whether a positive serology results correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. Methods. A neutralization assay was validated in a set of PCR confirmed positive specimens and in a negative cohort. 9,530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N=164) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and the levels of neutralizing antibodies detected was correlated. Neutralizing antibody titers (ID50) were also longitudinally monitored in SARS-CoV-2 PCR confirmed patients. Results. The SARS-CoV-2 neutralization assay had a PPA of 96.6% with a SARS-CoV-2 PCR test and a NPA of 98.0% across 100 negative controls. ID50 neutralization titers positively correlated with all three clinical serology platforms. Longitudinal monitoring of hospitalized PCR confirmed COVID-19 patients demonstrates they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2% and 78.4%, respectively. Conclusions. For the first time, we demonstrate that three widely available clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in COVID-19 patients. When a two-platform screen and confirm approach was used for SARS-CoV-2 serology, nearly 80% of two-platform positive specimens had neutralization titers (ID50 >50).

Background COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus that is responsible for the 2019 coronavirus pandemic. Acute infections should be diagnosed by polymerase chain reaction (PCR) based tests, but serology tests can demonstrate previous exposure to the virus. Methods We compared the performance of the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays using 179 negative subjects to determine negative percent agreement (NPA) and in 60 SARS-CoV-2 PCR confirmed positive patients to determine positive percent agreement (PPA) at three different timeframes following a positive SARS-CoV-2 PCR result. Results At ≥ 15 days, the PPA (95% CI) was 100 (86.3–100)% for the Diazyme IgM/IgG panel, 96.0 (79.7–99.9)% for the Roche total Ig assay, and 100 (86.3–100)% for the Abbott IgG assay. The NPA (95% CI) was 98.3 (95.2–99.7)% for the Diazyme IgM/IgG panel, 99.4 (96.9–100)% for the Roche total Ig assay, and 98.9 (96.0–99.9)% for the Abbott IgG assay. When the Roche total Ig assay was combined with either the Diazyme IgM/IgG panel or the Abbott IgG assay, the positive predictive value was 100% while the negative predictive value remained greater than 99%. Conclusions Our data demonstrates that the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays have similar clinical performance. We demonstrated a low false positive rate across all three platforms and observed that false positives observed on the Roche platform are unique compared to those observed on the Diazyme or Abbott assays. Using multiple platforms in tandem increases the PPVs which is important when screening populations with low disease prevalence.

Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast.

Background It is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. Methods A neutralization assay was validated in a set of PCR confirmed positive specimens and in a negative cohort. In addition, 9,530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (ID50) were also longitudinally monitored in SARS-CoV-2 PCR confirmed patients. Results The SARS-CoV-2 neutralization assay had a positive percent agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percent agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all three clinical serology platforms. Longitudinal monitoring of hospitalized PCR confirmed COVID-19 patients demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2% and 78.4%, respectively. Conclusions These three clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in COVID-19 patients. All SARS-CoV-2 PCR positive patients develop neutralizing antibodies.