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.

scholarly journals Two checkpoint complexes are independently recruited to sites of DNA damage in vivo

2001 ◽  
Vol 15 (21) ◽  
pp. 2809-2821 ◽  
Author(s):  
Justine A. Melo ◽  
Jen Cohen ◽  
David P. Toczyski
Keyword(s):  
Dna Damage ◽  
Living Cells ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Proteins ◽  
Checkpoint Protein ◽  
Checkpoint Adaptation ◽  
Replication Factors ◽  
Checkpoint Genes

The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by “sensing” DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1–GFP or Ddc2–GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2–GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.

scholarly journals The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast

2000 ◽  
Vol 14 (16) ◽  
pp. 2046-2059 ◽  
Author(s):  
Vera Paciotti ◽  
Michela Clerici ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Dna Damage ◽  
Dna Integrity ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
Dna Damaging Agents ◽  
Checkpoint Pathway ◽  
Functional Homolog

DDC2 is a novel component of the DNA integrity checkpoint pathway, which is required for proper checkpoint response to DNA damage and to incomplete DNA replication. Moreover, Ddc2 overproduction causes sensitivity to DNA-damaging agents and checkpoint defects. Ddc2 physically interacts with Mec1 and undergoes Mec1-dependent phosphorylation both in vitro and in vivo. The phosphorylation of Ddc2 takes place in late S phase and in G2 phase during an unperturbed cell cycle and is further increased in response to DNA damage. Because Ddc2 phosphorylation does not require any other known tested checkpoint factors but Mec1, the Ddc2–Mec1 complex might respond to the presence of some DNA structures independently of the other known checkpoint proteins. Our findings suggest that Ddc2 may be the functional homolog of Schizosaccharomyces pombe Rad26, strengthening the hypothesis that the mechanisms leading to checkpoint activation are conserved throughout evolution.

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 Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences

2006 ◽  
Vol 26 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Guochun Jiang ◽  
Aziz Sancar
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Excision Repair ◽  
Dna Lesions ◽  
Human Cells ◽  
Dna Damage Checkpoint ◽  
Damage Site ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
Transcription Complexes

ABSTRACT We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.

scholarly journals The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Takeshi Wakida ◽  
Masae Ikura ◽  
Kenji Kuriya ◽  
Shinji Ito ◽  
Yoshiharu Shiroiwa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Proliferating Cells ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Promote Cell Proliferation

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

scholarly journals DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

2002 ◽  
Vol 158 (5) ◽  
pp. 863-872 ◽  
Author(s):  
Matthew P. Stokes ◽  
Ruth Van Hatten ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Alkylating Agents ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Damaged Dna

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

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.

Checkpoint Proteins Influence Telomeric Silencing and Length Maintenance in Budding Yeast

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1577-1591 ◽  
Author(s):  
Maria Pia Longhese ◽  
Vera Paciotti ◽  
Holger Neecke ◽  
Giovanna Lucchini
Keyword(s):  
Dna Damage ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Dna Damage Checkpoint ◽  
Genetic Integrity ◽  
Cycle Progression ◽  
Telomere Shortening ◽  
Checkpoint Proteins ◽  
Sir Proteins ◽  
Synthetic Capacity

AbstractA complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity. In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism. We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity. In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres. Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size. The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways. The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53.

scholarly journals Fission Yeast Rad17 Associates with Chromatin in Response to Aberrant Genomic Structures

2001 ◽  
Vol 21 (10) ◽  
pp. 3289-3301 ◽  
Author(s):  
Mihoko Kai ◽  
Hiroyuki Tanaka ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Genomic Structure ◽  
Small Subunit ◽  
S Phase ◽  
Dna Integrity ◽  
Checkpoint Proteins ◽  
Defective Mutant ◽  
Mutant Cells

ABSTRACT Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Δ or rad3Δ cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Δ are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys118 of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in therad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.

Regulation of Septum Formation in Aspergillus nidulans by a DNA Damage Checkpoint Pathway

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1055-1067
Author(s):  
Steven D Harris ◽  
Peter R Kraus
Keyword(s):  
Dna Damage ◽  
Aspergillus Nidulans ◽  
Cellular Response ◽  
Dna Damage Checkpoint ◽  
Temperature Sensitive ◽  
Dna Metabolism ◽  
Chromosomal Dna ◽  
Checkpoint Response ◽  
Septum Formation ◽  
Checkpoint Pathway

Abstract In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.

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