Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication [microarray]
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ABSTRACT:
PROVIDER: PRJNA562850 | ENA |
REPOSITORIES: ENA
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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:Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication
| PRJNA562848 | ENA
Project description:Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication [sequencing]
| PRJNA562847 | ENA
Project description:Intracellular levels of deoxyribonucleoside triphosphate (dNTP) must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools have recently been associated with increased mutagenesis, genomic instability and tumorigenesis. However, the mechanisms by which low or imbalanced dNTP pools affect DNA replication remain poorly understood. Here, we have modulated the activity of ribonucleotide reductase (RNR), a key enzyme catalyzing a rate-limiting step of dNTP production, to monitor the effect of altered dNTP levels on replication dynamics in budding yeast. We show that dNTP pools are limiting for normal DNA synthesis as upregulation of RNR activity increases replication fork speed. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition from a regular- to a slow-replication mode within minutes after S-phase entry. Interestingly, we found that upregulation of RNR activity delays this transition and that dNTP levels modulate both fork speed and origin usage under replication stress. Moreover, we report that chromosomal instability (CIN) mutants show increased dNTP pools and enhanced DNA synthesis in the presence of HU. Since upregulation of RNR allows forks to progress faster in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.
2012-01-01 | GSE33686 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2020-03-17 | GSE136605 | GEO
Project description:Mannose is an anti-cancer sugar that inhibits cell proliferation and enhances chemotherapy. How mannose exerts its anti-cancer activities, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced a global metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphate (dNTP) pools. This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, a pharmacological inhibition of de novo dNTP biosynthesis was sufficient to sensitize the non-engineered cells to cisplatin and inhibit dormant origin firing, suggesting the dNTP loss-induced genome instability as a major mechanism for the anti-cancer activities of mannose.
2023-07-06 | PXD036449 | Pride
Project description:Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication and genome stability. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may constitute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication
2017-02-13 | GSE90054 | GEO
Project description:Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here we show that CDK-induced replication stress is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, Mus81- dependent DNA damage, and to genome instability. Cells respond to this replication stress by increasing dNTP supply through Set2-dependent MBFinduced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through loss of Set2-dependent H3K36 tri-methylation, results in further dNTP depletion due to reduced RNR expression, leading to replication catastrophe and cell death. This lethality can be rescued by increasing dNTP levels. Similarly, we find loss of the DNA integrity checkpoint results in synthetic lethality with Wee1 inactivation, which is also associated with replication collapse through critically low dNTP levels. Together, these findings support a ‘dNTP supply and demand’ model in which maintaining dNTP homeostasis is essential in preventing replication catastrophe in response to CDK-induced replication stress.
2018-05-26 | GSE113747 | GEO
Project description:Upon replication stress, the Mec1ATR kinase triggers the downregulation of transcription, reducing the level of RNA polymerase on chromatin to facilitate replication fork progression. We identify a hydroxyurea-induced phosphorylation site at Mec1-S1991 that contributes to the eviction of RNAPII and RNAPIII during replication stress. The non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII with a Rpb3-TAP fusion, suggesting that lethality arises from replication-transcription conflicts. Coincident with a failure to repress gene expression, highly transcribed genes like GAL1 persist at nuclear pores in mec1-S1991A cells. Consistently, we find pore proteins and several components controlling RNAPII and RNAPIII transcription are phosphorylated in a Mec1-dependent manner, suggesting that Mec1-S1991 phosphorylation limits conflicts between replication and either RNA polymerase complex. We further show that Mec1 contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase.
2021-09-08 | GSE180164 | GEO