scholarly journals Disease-associated DNA2 nuclease–helicase protects cells from lethal chromosome under-replication

2020 ◽  
Author(s):  
Benoît Falquet ◽  
Gizem Ölmezer ◽  
Franz Enkner ◽  
Dominique Klein ◽  
Kiran Challa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Dna Damage Checkpoint ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Essential Function ◽  
Mutant Cells ◽  
Lagging Strand

Abstract DNA2 is an essential nuclease–helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5′-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5′-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2’s role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1’s ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2’s role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.

53BP1: function and mechanisms of focal recruitment

2009 ◽  
Vol 37 (4) ◽  
pp. 897-904 ◽  
Author(s):  
Jennifer E. FitzGerald ◽  
Muriel Grenon ◽  
Noel F. Lowndes
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Damage Response ◽  
Nuclear Structures ◽  
Genotoxic Insult ◽  
Covalent Histone Modifications

53BP1 (p53-binding protein 1) is classified as a mediator/adaptor of the DNA-damage response, and is recruited to nuclear structures termed foci following genotoxic insult. In the present paper, we review the functions of 53BP1 in DNA-damage checkpoint activation and DNA repair, and the mechanisms of its recruitment and activation following DNA damage. We focus in particular on the role of covalent histone modifications in this process.

scholarly journals Phosphorylation of the Budding Yeast 9-1-1 Complex Is Required for Dpb11 Function in the Full Activation of the UV-Induced DNA Damage Checkpoint

2008 ◽  
Vol 28 (15) ◽  
pp. 4782-4793 ◽  
Author(s):  
Fabio Puddu ◽  
Magda Granata ◽  
Lisa Di Nola ◽  
Alessia Balestrini ◽  
Gabriele Piergiovanni ◽  
...  
Keyword(s):  
Dna Damage ◽  
Budding Yeast ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
M Phase ◽  
Mutant Cells ◽  
Full Activation

ABSTRACT Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G1 phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Δ dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.

The Recql5 Helicase Is a Novel Downstream Target of Jak2V617F and Maintains Genome Stability in Response to Replication Stress

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 822-822
Author(s):  
Edwin Chen ◽  
Jong-Sook Ahn ◽  
Lawrence J Breyfogle ◽  
Anthony R Green ◽  
Benjamin L. Ebert ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Replication Stress ◽  
Therapeutic Window ◽  
Replication Forks ◽  
Erythroid Progenitor ◽  
Stalled Replication Forks

Abstract The JAK2V617F mutation is present in a majority of patients with chronic myeloproliferative neoplasms (MPNs). Mutant JAK2 induces hyperactivation of multiple downstream signaling processes with the net effect of conferring cells with a pro-survival advantage. In particular, JAK2V617F-expressing cells tolerate increased DNA damage and higher levels of intracellular reactive oxygen species. However, the mechanisms by which increased genotoxic tolerance is mediated remain unclear. Previously, we performed gene expression analysis on autologous wildtype and JAK2V617F-heterozygous erythroblasts from 36 MPN patients, and observed increased expression of the RECQL5 helicase in JAK2-mutant erythroblasts. Increased Recql5 transcript and protein levels were also validated in Hoxb8-immortalized, GMP-like cell lines derived from wildtype and Jak2V617F knock-in mice (WT-B8 and VF-B8 cells, respectively). Recql5 up-regulation was dependent on the Pi3k-Akt pathway, and was independent of Stat1/5 and Mapk/Erk activity. As the Recql family of helicases plays a critical role in replication fork stability, we tested whether Recql5 could modulate sensitivity of JAK2-expressing cells to agents which promote replication stress, such as hydroxyurea (HU) and aphidicolin (APH). Strikingly, VF-B8 cells transduced with two different Recql5 shRNAs were more susceptible to HU- and APH-induced apoptosis when compared to Recql5-deficient WT-B8 cells. Replication stress-induced cytotoxicity was accompanied by increased gamma-H2Ax-marked double stranded breaks (DSBs) and activation of DNA repair pathways. Importantly, re-introduction of an shRNA-resistant Recql5 cDNA successfully rescued Recql5-deficient VF-B8 cells from HU- and APH-cytotoxicity. Molecularly, we show that Recql5 plays two roles to protect against DSB formation and cell death in Jak2-mutant cells. First, we visualized replication tracts on individual DNA fibers by chromosome combing and observed that Recql5-deficient VF-B8 cells treated with HU exhibit increased numbers of stalled replication forks. Moreover, Recql5 deficiency also led to an inability to restart forks stalled by HU treatment. This indicates that the absence of Recql5 leads to replication forks which are unstably arrested upon HU treatment, leading to fork collapse and the generation of DSBs. Second, we quantified the rate of single-stranded annealing (SSA) repair following Recql5 knockdown. Consistent with previous reports, we observed increased rates of SSA repair in VF-B8 cells compared to WT-B8 cells. However, this difference in the rate of SSA repair is abrogated upon Recql5 knockdown, suggesting that Recql5 functions as a mediator for the SSA DNA repair pathway. Cumulatively, these findings suggest that Recql5 up-regulation in Jak2V617F-expressing cells plays a role in protecting against DNA damage-induced cell death through (1) stabilization of stalled replication forks thus preventing their collapse, and (2) promoting rapid (albeit error prone) SSA DNA repair to ameliorate genomic instability. Finally, we tested whether modulation of RECQL5 could also increase sensitivity of JAK2V617F-positive cells from primary MPN patients to HU. Following depletion with RECQL5, c-kit-enriched peripheral blood mononuclear cells from 2 essential thrombocythemia and 3 myelofibrosis patients were grown in semi-solid medium supplemented with HU for 14 days. Strikingly, we observed more specific eradication of JAK2V617F-positive erythroid progenitor colonies compared to autologous wildtype colonies from all patients examined. In contrast, no specific killing of JAK2V617F-positive erythroblasts was seen following transduction of control hairpins. This suggests that RECQL5 knockdown may potentially open a therapeutic window by sensitizing Jak2V617F-expressing cells to HU and other agents that induce replication stress. Disclosures No relevant conflicts of interest to declare.

scholarly journals RAD9, RAD17, and RAD24 Are Required for S Phase Regulation in Saccharomyces cerevisiae in Response to DNA Damage

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 45-62 ◽  
Author(s):  
A G Paulovich ◽  
R U Margulies ◽  
B M Garvik ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Repair ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Alkylation Damage ◽  
Checkpoint Pathway ◽  
Phase Regulation ◽  
Phase Progression

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9Δ, rad17Δ, and rad24Δ. Furthermore, the triple rad9Δ rad17Δ rad24Δ mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1Δ, mag1Δ, apn1Δ, rev3Δ, rad18Δ, rad16Δ, dun1-Δ100, sad4-1, tel1Δ, rad26Δ, rad51Δ, rad52-1, rad54Δ, rad14Δ, rad1Δ, pol30–46, pol30–52, mad3Δ, pds1Δ/esp2Δ, pms1Δ, mlh1Δ, and msh2Δ are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.

scholarly journals Slx4 Regulates DNA Damage Checkpoint-dependent Phosphorylation of the BRCT Domain Protein Rtt107/Esc4

2006 ◽  
Vol 17 (1) ◽  
pp. 539-548 ◽  
Author(s):  
Tania M. Roberts ◽  
Michael S. Kobor ◽  
Suzanne A. Bastin-Shanower ◽  
Miki Ii ◽  
Sonja A. Horte ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Alkylation Damage ◽  
Binding Partner ◽  
Replication Restart ◽  
Damage Response ◽  
Domain Protein

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107Δ, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.

scholarly journals Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Christophe de La Roche Saint-André ◽  
Vincent Géli
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genetic Material ◽  
S Phase ◽  
Replication Forks ◽  
H3k4 Methylation ◽  
Origin Firing ◽  
Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

Evaluation of DNA Damage, DNA Repair, Oxidative Stress, and Cell Death Caused by Helicobacter pylori in Human Adenocarcinoma Cells

2021 ◽  
Author(s):  
DİDEM ORAL ◽  
ÜNZİLE SUR ◽  
gizem özkemahlı ◽  
Anıl Yirüna ◽  
N. DİLARA ZEYBEK ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Helicobacter Pylori ◽  
Adenocarcinoma Cells
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