scholarly journals Basal CHK1 activity safeguards its stability to maintain intrinsic S-phase checkpoint functions

2019 ◽  
Vol 218 (9) ◽  
pp. 2865-2875 ◽  
Author(s):  
Jone Michelena ◽  
Marco Gatti ◽  
Federico Teloni ◽  
Ralph Imhof ◽  
Matthias Altmeyer
Keyword(s):  
Cell Proliferation ◽  
Steady State ◽  
Cell Survival ◽  
Replication Fork ◽  
S Phase ◽  
Genome Integrity ◽  
Replication Machinery ◽  
Replication Fork Progression ◽  
Steady State Activity ◽  
S Phase Checkpoint

The DNA replication machinery frequently encounters impediments that slow replication fork progression and threaten timely and error-free replication. The CHK1 protein kinase is essential to deal with replication stress (RS) and ensure genome integrity and cell survival, yet how basal levels and activity of CHK1 are maintained under physiological, unstressed conditions is not well understood. Here, we reveal that CHK1 stability is controlled by its steady-state activity during unchallenged cell proliferation. This autoactivatory mechanism, which depends on ATR and its coactivator ETAA1 and is tightly associated with CHK1 autophosphorylation at S296, counters CHK1 ubiquitylation and proteasomal degradation, thereby preventing attenuation of S-phase checkpoint functions and a compromised capacity to respond to RS. Based on these findings, we propose that steady-state CHK1 activity safeguards its stability to maintain intrinsic checkpoint functions and ensure genome integrity and cell survival.

scholarly journals The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

2005 ◽  
Vol 168 (7) ◽  
pp. 999-1012 ◽  
Author(s):  
Jeff Bachant ◽  
Shannon R. Jessen ◽  
Sarah E. Kavanaugh ◽  
Candida S. Fielding
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
S Phase ◽  
Origin Of Replication ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Hydroxyurea Treatment ◽  
Chromatid Cohesion ◽  
S Phase Checkpoint

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

scholarly journals Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities

2004 ◽  
Vol 24 (8) ◽  
pp. 3198-3212 ◽  
Author(s):  
Jorge Z. Torres ◽  
Sandra L. Schnakenberg ◽  
Virginia A. Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Replication Fork ◽  
Opportunity To Learn ◽  
Normal Growth ◽  
Dna Helicase ◽  
S Phase ◽  
Exogenous Dna ◽  
Fork Stalling ◽  
S Phase Checkpoint

ABSTRACT Rrm3p is a 5′-to-3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

scholarly journals Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
André Franz ◽  
Paul A. Pirson ◽  
Domenic Pilger ◽  
Swagata Halder ◽  
Divya Achuthankutty ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dynamic Process ◽  
Metabolic Pathways ◽  
Replication Fork ◽  
Genome Instability ◽  
Genome Integrity ◽  
Substrate Selection ◽  
Replication Fork Progression ◽  
Replication Factors ◽  
Spatiotemporal Control

Abstract The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

Interaction between the p53 genotype of lung cancer and response to mTOR inhibitor combined with irradiation therapy

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22028-e22028
Author(s):  
Y. Nagata ◽  
T. Tojo ◽  
K. Ohnishi ◽  
A. Takahashi ◽  
T. Ohnishi ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Survival ◽  
Cell Cycle Arrest ◽  
Mtor Inhibitor ◽  
P53 Gene ◽  
S Phase ◽  
Cycle Arrest ◽  
Irradiation Therapy

e22028 Background: Frequent activation of the PI3K/Akt/mTOR pathway and aberrations of tumor suppressor gene p53 are associated with therapeutic resistance in lung cancer. Nevertheless, the possibility of the variants of p53 genotype to affect response to mTOR inhibitor combined with irradiation therapy remains still unclear. Methods: Human non-small lung cancer cell line H1299 with p53 null genotype, was transfected with wild type or mutated p53 gene (H1299/wtp53 (WT), H1299/mp53 (MT)). Both cell survival and cell proliferation were estimated by colony formation assay to assess differences between WT and MT in sensitivity to rapamycin and ability of rapamycin to enhance radiation sensitivity. Cells were treated according to the individual study; DMSO (control), rapamycin (100 nM for 1 hour), irradiation (IR) (increasing doses), combination (RR) (rapamycin followed by irradiation). Changes in the cell cycle were also analyzed by flow cytometry. Results: Rapamycin decreased cell survival only in WT (P < 0.01, vs. control). MT was resistant to rapamycin exhibiting slightly inhibited cell proliferation. Compared with IR, RR with no less than 6 Gy radiation enhanced inhibitory effects on both cell survival and proliferation independent of p53 genotype (P < 0.01 in WT and P < 0.01 in MT, respectively), that indicating additive interaction. Cell cycle analysis demonstrated rapamycin induced G1 cell cycle arrest in both types of cells compared with controls (P < 0.01 in WT and P < 0.05 in MT, respectively) at 24 hours after treatment. Enhancement of G1 arrest by RR was observed in both WT (P < 0.01, vs. IR) and MT (P < 0.01, vs. IR) at the same time point. In addition, RR caused a greater reduction of cells in S phase than that of IR regardless of p53 gene status (P < 0.01 in WT and P < 0.01 in MT, respectively). Conclusions: Rapamycin is an effective radiosensitizer in a p53 independent manner, in which increased G1 cell cycle arrest and decrease in S phase cells are responsible for increased growth inhibitory effect. It will enable us to provide more appropriate treatment to patients with mutated p53 gene. No significant financial relationships to disclose.

scholarly journals Chk1 Requirement for High Global Rates of Replication Fork Progression during Normal Vertebrate S Phase

2006 ◽  
Vol 26 (8) ◽  
pp. 3319-3326 ◽  
Author(s):  
Eva Petermann ◽  
Apolinar Maya-Mendoza ◽  
George Zachos ◽  
David A. F. Gillespie ◽  
Dean A. Jackson ◽  
...  
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Fork Progression ◽  
Chk1 Inhibitor ◽  
Interfering Rna ◽  
First Time ◽  
Dt40 Cells

ABSTRACT Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1 −/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1 −/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3 −/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1 −/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1 −/− cells are associated with the accumulation of aberrant replication fork structures.

scholarly journals Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression

2013 ◽  
Vol 6 (1) ◽  
Author(s):  
Srividya Bhaskara ◽  
Vincent Jacques ◽  
James R Rusche ◽  
Eric N Olson ◽  
Bradley R Cairns ◽  
...  
Keyword(s):  
Dna Replication ◽  
Histone Deacetylases ◽  
Replication Fork ◽  
S Phase ◽  
Replication Fork Progression

scholarly journals Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
Author(s):  
Ling Yin ◽  
Alexandra Monica Locovei ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Damage Checkpoint ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Fork Stalling ◽  
S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

scholarly journals The CMG helicase bypasses DNA protein cross-links to facilitate their repair

2018 ◽  
Author(s):  
Justin L. Sparks ◽  
Alan O. Gao ◽  
Markus Räschle ◽  
Nicolai B. Larsen ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Cross Link ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Cross Links ◽  
Leading Strand ◽  
Nucleoprotein Complexes ◽  
Lagging Strand

SummaryCovalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. UsingXenopus laevisegg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisome’s response to nucleoprotein barriers.

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