Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analysis of the tolerance to DNA alkylating damage in MEC1 and RAD53 checkpoint mutants of Saccharomyces cerevisiae.

ABSTRACT: Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1? or rad53? mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.

SUBMITTER: Gallego-Sanchez A

PROVIDER: S-EPMC3834268 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Analysis of the tolerance to DNA alkylating damage in MEC1 and RAD53 checkpoint mutants of Saccharomyces cerevisiae.

Gallego-Sánchez Alfonso A Ufano Sandra S Andrés Sonia S Bueno Avelino A

PloS one 20131119 11

Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches u ...[more]

PMID: 24260543

Similar Datasets

The conserved Mec1/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in Saccharomyces cerevisiae.

Project description:How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the approximately 50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions.

| S-EPMC1142443 | biostudies-literature

Rpd3L Contributes to the DNA Damage Sensitivity of Saccharomyces cerevisiae Checkpoint Mutants.

Project description:DNA replication forks that are stalled by DNA damage activate an S-phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Using a whole-genome sequencing approach, we identified two additional genes, RXT2 and RPH1, whose mutation can also partially suppress this DNA damage sensitivity. We provide evidence that RXT2 and RPH1 act in a common pathway, which is distinct from the EXO1 pathway. Analysis of additional mutants indicates that suppression works through the loss of the Rpd3L histone deacetylase complex. Our results suggest that the loss or absence of histone acetylation, perhaps at stalled forks, may contribute to cell death in the absence of a functional checkpoint.

| S-EPMC6366903 | biostudies-literature

DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

Project description:Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

| S-EPMC3664849 | biostudies-literature

Rpd3L Contributes to the DNA Damage Sensitivity of Saccharomyces cerevisiae Checkpoint Mutants

Project description:DNA replication forks that are stalled by DNA damage activate an S phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Here,we identified that loss of the histone deacetylase complex Rpd3L promotes survival of rad53∆ cells exposed to DNA damaging agents. From epistasis analysis, we show that this suppression operates in a separate pathway from the previously described suppression by deletion of EXO1.

2019-02-27 | GSE113869 | GEO

The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling.

Project description:Double-strand breaks (DSBs) elicit a DNA damage response, resulting in checkpoint-mediated cell-cycle delay and DNA repair. The Saccharomyces cerevisiae Sae2 protein is known to act together with the MRX complex in meiotic DSB processing, as well as in DNA damage response during the mitotic cell cycle. Here, we report that cells lacking Sae2 fail to turn off both Mec1- and Tel1-dependent checkpoints activated by a single irreparable DSB, and delay Mre11 foci disassembly at DNA breaks, indicating that Sae2 may negatively regulate checkpoint signalling by modulating MRX association at damaged DNA. Consistently, high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1-dependent Sae2 phosphorylation is necessary for these Sae2 functions, suggesting that the two kinases, once activated, may regulate checkpoint switch off through Sae2-mediated inhibition of MRX signalling.

| S-EPMC1369247 | biostudies-literature

The DNA Damage Checkpoint and the Spindle Position Checkpoint Maintain Meiotic Commitment in Saccharomyces cerevisiae.

Project description:During meiosis, diploid progenitor cells undergo one round of DNA replication followed by two rounds of chromosome segregation to form haploid gametes. Once cells initiate the meiotic divisions, it is imperative that they finish meiosis. A failure to maintain meiosis can result in highly aberrant polyploid cells, which could lead to oncogenesis in the germline. How cells stay committed to finishing meiosis, even in the presence of a mitosis-inducing signal, is poorly understood. We addressed this question in budding yeast, in which cells enter meiosis when starved. If nutrient-rich medium is added before a defined commitment point in mid-prometaphase I, they can return to mitosis. Cells in stages beyond the commitment point will finish meiosis, even with nutrient addition. Because checkpoints are signaling pathways known to couple cell-cycle processes with one another, we asked if checkpoints could ensure meiotic commitment. We find that two checkpoints with well-defined functions in mitosis, the DNA damage checkpoint and the spindle position checkpoint, have crucial roles in meiotic commitment. With nutrient-rich medium addition at stages beyond the commitment point, cells that are deficient in both checkpoints because they lack Rad53 and either Bub2, Bfa1, or Kin4 can return to mitotic growth and go on to form polyploid cells. The results demonstrate that the two checkpoints prevent cells from exiting meiosis in the presence of a mitosis-inducing signal. This study reveals a previously unknown function for the DNA damage checkpoint and the spindle position checkpoint in maintaining meiotic commitment.

| S-EPMC6363850 | biostudies-literature

The Yeast DNA Damage Checkpoint Kinase Rad53 Targets the Exoribonuclease, Xrn1.

Project description:The highly conserved DNA damage response (DDR) pathway monitors the genomic integrity of the cell and protects against genotoxic stresses. The apical kinases, Mec1 and Tel1 (ATR and ATM in human, respectively), initiate the DNA damage signaling cascade through the effector kinases, Rad53 and Chk1, to regulate a variety of cellular processes including cell cycle progression, DNA damage repair, chromatin remodeling, and transcription. The DDR also regulates other cellular pathways, but direct substrates and mechanisms are still lacking. Using a mass spectrometry-based phosphoproteomic screen in Saccharomyces cerevisiae, we identified novel targets of Rad53, many of which are proteins that are involved in RNA metabolism. Of the 33 novel substrates identified, we verified that 12 are directly phosphorylated by Rad53 in vitro: Xrn1, Gcd11, Rps7b, Ded1, Cho2, Pus1, Hst1, Srv2, Set3, Snu23, Alb1, and Scp160. We further characterized Xrn1, a highly conserved 5' exoribonuclease that functions in RNA degradation and the most enriched in our phosphoproteomics screen. Phosphorylation of Xrn1 by Rad53 does not appear to affect Xrn1's intrinsic nuclease activity in vitro, but may affect its activity or specificity in vivo.

| S-EPMC6288840 | biostudies-other

The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint.

Project description:Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1.

| S-EPMC1850967 | biostudies-literature

The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53.

Project description:In this study, we mutated autophosphorylation sites in Rad53 based on their conservation with previously identified autophosphorylation sites in the mammalian Rad53 ortholog, Chk2. As with wild-type Rad53, the autophosphorylation mutant, rad53-TA, undergoes Mec1/Tel1-dependent interactions with Rad9 and Dun1 in response to genotoxic stress. Whereas rad53-TA in vitro kinase activity is severely impaired, the rad53-TA strains are not completely deficient for cell-cycle checkpoint functions, indicating that the mutant kinase retains a basal level of function. We describe a genetic interaction among Rad53, Dun1, and the 14-3-3 proteins Bmh1 and Bmh2 and present evidence that 14-3-3 proteins directly facilitate Rad53 function in vivo. The data presented account for the previously observed checkpoint defects associated with 14-3-3 mutants in Saccharomyces pombe and Saccharomyces cerevisiae. The 14-3-3 functional interaction appears to modulate Rad53 activity, reminiscent of 14-3-3's effect on human Raf1 kinase and distinct from the indirect mode of regulation by 14-3-3 observed for Chk1 or Cdc25.

| S-EPMC1797148 | biostudies-literature

Loss of SOD1 and LYS7 sensitizes Saccharomyces cerevisiae to hydroxyurea and DNA damage agents and downregulates MEC1 pathway effectors.

Project description:Aerobic metabolism produces reactive oxygen species, including superoxide anions, which cause DNA damage unless removed by scavengers such as superoxide dismutases. We show that loss of the Cu,Zn-dependent superoxide dismutase, SOD1, or its copper chaperone, LYS7, confers oxygen-dependent sensitivity to replication arrest and DNA damage in Saccharomyces cerevisiae. We also find that sod1Delta strains, and to a lesser extent lys7Delta strains, when arrested with hydroxyurea (HU) show reduced induction of the MEC1 pathway effector Rnr3p and of Hug1p. The HU sensitivity of sod1Delta and lys7Delta strains is suppressed by overexpression of TKL1, a transketolase that generates NADPH, which balances redox in the cell and is required for ribonucleotide reductase activity. Our results suggest that the MEC1 pathway in sod1Delta mutant strains is sensitive to the altered cellular redox state due to increased superoxide anions and establish a new relationship between SOD1, LYS7, and the MEC1-mediated checkpoint response to replication arrest and DNA damage in S. cerevisiae.

| S-EPMC1291217 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data