Phosphoproteomic Datasets for Mec1/ATR signaling in Checkpoint-deficient (rad53Δ) cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
Bokun Xie
LAB HEAD: Marcus Smolka
PROVIDER: PXD051892 | Pride | 2024-06-05
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 072520_EJS_R53delvsWT_H1.mzXML | Mzxml | |||
| 072520_EJS_R53delvsWT_H1.pep.xml | Pepxml | |||
| 072520_EJS_R53delvsWT_H1.raw | Raw | |||
| 072520_EJS_R53delvsWT_H10.mzXML | Mzxml | |||
| 072520_EJS_R53delvsWT_H10.pep.xml | Pepxml |
Items per page: 5 1 - 5 of 90 |
Similar Datasets
Project description:The Mec1/ATR kinase is crucial for genome maintenance, although the mechanisms by which it controls DNA repair pathways remain elusive. Here we uncovered a role for Mec1/ATR in controlling homologous recombination (HR) factors in response to hyper-resection of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit Mec1-dependent hyper-phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11.
2021-03-10 | PXD017286 | Pride
Project description:The Mec1/ATR kinase is crucial for genome maintenance, although the mechanisms by which it controls DNA repair pathways remain elusive. Here we uncovered a role for Mec1/ATR in controlling homologous recombination (HR) factors in response to hyper-resection of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit Mec1-dependent hyper-phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11.
2021-03-10 | PXD023438 | Pride
Project description:The Mec1/ATR kinase is crucial for genome maintenance, although the mechanisms by which it controls DNA repair pathways remain elusive. Here we uncovered a role for Mec1/ATR in controlling homologous recombination (HR) factors in response to hyper-resection of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit Mec1-dependent hyper-phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 promotes recruitment of STR to the 9-1-1 clamp via Dpb11 to regulate recombinogenic processes.
2021-03-10 | PXD017289 | Pride
Project description:The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by the Mec1 kinase and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 phosphorylation and activation has been well documented, less is known about the processes that counteract its kinase activity during the response to a DNA break. Here, we describe that PP4 dephosphorylates Rad53 during the repair of a DNA lesion, a process that affects the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection in a process that relies mainly on Sgs1/Dna2. Consequently, elimination of PP4 activity affects DNA resection and repair by single-strand annealing, defects that are bypassed by reducing the hyper-phosphorylation state of Rad53 observed in the absence of the phosphatase. These results confirms that Rad53 is one of the principal targets of PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational repair pathways that depend on long-range resection for its execution.
2019-09-12 | PXD013782 | Pride
Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.
2014-11-20 | GSE63444 | GEO
Project description:The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1–Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because dNTP levels present in G1 phase are not sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use dNTP shortage to detect the onset of DNA replication and activate the Mec1–Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
2020-03-17 | GSE136602 | GEO
Project description:The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1–Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because dNTP levels present in G1 phase are not sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use dNTP shortage to detect the onset of DNA replication and activate the Mec1–Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
2020-03-17 | GSE136601 | GEO
Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. Strand-specific transcriptome analysis of biological replicates of WT cells (JKM139 strain) at T0, or 60 and 240 minutes after HO induction, and of xrn1∆, rrp6∆ and trf4∆ cells at T0.
2014-11-20 | E-GEOD-63444 | biostudies-arrayexpress
Project description:The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we employed a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of “free” Mec1 Activation Domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator-proteins, “free” MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1’s role in genome maintenance is separable from a previously unappreciated pro-replicative function. Both Mec1’s functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.
2019-01-14 | PXD009734 | Pride
Project description:The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load. The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load.
2019-01-12 | GSE124948 | GEO