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 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 Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

2003 ◽  
Vol 23 (4) ◽  
pp. 1441-1452 ◽  
Author(s):  
Vladimir I. Bashkirov ◽  
Elena V. Bashkirova ◽  
Edwin Haghnazari ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Dna Damage ◽  
Target Genes ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Kinase Signaling ◽  
M Cell ◽  
Fha Domain ◽  
Checkpoint Pathway

ABSTRACT The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G2/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G2/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

scholarly journals The Synergistic Anticancer Effect of a Combination of Chidamide and Etoposide Against NK/T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3987-3987
Author(s):  
Wenting Song ◽  
Zhan Chen ◽  
Cunzhen Shi ◽  
Yuyang Gao ◽  
Xiaoyan Feng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
T Cell ◽  
Histone Acetylation ◽  
Hematological Malignancies ◽  
Cell Lymphoma ◽  
Dna Damaging Agents ◽  
Etoposide Treatment

Abstract Natural killer/T cell lymphoma (NKTCL) is a highly aggressive hematological malignancy. However, there is currently no consensus on first-line therapies for refractory/relapsed patients. Chidamide is a self-researched and developed HDACs inhibitor, and when combined with DNA-damaging agents, exhibited a clinical synergistic effect for the treatment of some solid tumors and hematological malignancies. Thus in this study, a series of in vitro and in vivo experiments were conducted to explore the efficacy and potential mechanisms of combined chidamide and etoposide treatment in NKTCL. We demonstrated that chidamide or etoposide alone dose- and time-dependently inhibited the cell viability of NKTCL cell lines, YT, NKYS and KHYG-1. Functional experiments suggested that combined chidamide and etoposide treatment exerted synergistic antiproliferation effect and enhanced cell apoptotic death both in vitro and in vivo. Furthermore, the expression of DNA damage related proteins was detected and we also examined the alternations in histone acetylation, cell cycle progression, and mitochondrial membrane potential (MMP). The results suggested that increased histone acetylation, cell cycle arrest at the G2/M phase and loss of MMP, converging to greater DNA damage, might account for the synergism of the combination of chidamide and etoposide in NKTCL. Taken together, our study supplements the clinical application of combining HDACs inhibitors and DNA-damaging agents on treating hematological malignancies but also provide an experimental basis for improved therapeutic efficacy and decreased complications for patients with NKTCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Nuclear Accumulation of LAP1:TRF2 Complex during DNA Damage Response Uncovers a Novel Role for LAP1

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1804
Author(s):  
Cátia D. Pereira ◽  
Filipa Martins ◽  
Mariana Santos ◽  
Thorsten Müeller ◽  
Odete A. B. da Cruz e Silva ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Nuclear Accumulation ◽  
Protein Levels ◽  
Dna Damaging Agents ◽  
Physiological Properties ◽  
Damage Response

Lamina-associated polypeptide 1 (LAP1) is a nuclear envelope (NE) protein whose function remains poorly characterized. In a recent LAP1 protein interactome study, a putative regulatory role in the DNA damage response (DDR) has emerged and telomeric repeat-binding factor 2 (TRF2), a protein intimately associated with this signaling pathway, was among the list of LAP1 interactors. To gain insights into LAP1′s physiological properties, the interaction with TRF2 in human cells exposed to DNA-damaging agents was investigated. The direct LAP1:TRF2 binding was validated in vitro by blot overlay and in vivo by co-immunoprecipitation after hydrogen peroxide and bleomycin treatments. The regulation of this protein interaction by LAP1 phosphorylation was demonstrated by co-immunoprecipitation and mass spectrometry following okadaic acid exposure. The involvement of LAP1 and TRF2 in the DDR was confirmed by their increased nuclear protein levels after bleomycin treatment, evaluated by immunoblotting, as well as by their co-localization with DDR factors at the NE and within the nucleoplasm, assessed by immunocytochemistry. Effectively, we showed that the LAP1:TRF2 complex is established during a cellular response against DNA damage. This work proposes a novel functional role for LAP1 in the DDR, revealing a potential biological mechanism that may be disrupted in LAP1-associated pathologies.

scholarly journals TP5, a Peptide Inhibitor of Aberrant and Hyperactive CDK5/p25: A Novel Therapeutic Approach against Glioblastoma

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1935
Author(s):  
Emeline Tabouret ◽  
Herui Wang ◽  
Niranjana Amin ◽  
Jinkyu Jung ◽  
Romain Appay ◽  
...  
Keyword(s):  
Dna Damage ◽  
Therapeutic Option ◽  
Selective Inhibition ◽  
G1 Arrest ◽  
Cyclin Dependent Kinase ◽  
Dna Damaging Agents ◽  
Induced Apoptosis ◽  
Cyclin Dependent Kinase 5

We examined the efficacy of selective inhibition of cyclin-dependent kinase 5 (CDK5) in glioblastoma by TP5. We analyzed its impact in vitro on CDK5 expression and activity, cell survival, apoptosis and cell cycle. DNA damage was analyzed using the expression of γH2A.X and phosphorylated ATM. Its tolerance and efficacy were assessed on in vivo xenograft mouse models. We showed that TP5 decreased the activity but not the expression of CDK5 and p35. TP5 alone impaired cell viability and colony formation of glioblastoma cell lines and induced apoptosis. TP5 increased DNA damage by inhibiting the phosphorylation of ATM, leading to G1 arrest. Whereas CDK5 activity is increased by DNA-damaging agents such as temozolomide and irradiation, TP5 was synergistic with either temozolomide or irradiation due to an accumulation of DNA damage. Concomitant use of TP5 and either temozolomide or irradiation reduced the phosphorylation of ATM, increased DNA damage, and inhibited the G2/M arrest induced by temozolomide or irradiation. TP5 alone suppressed the tumor growth of orthotopic glioblastoma mouse model. The treatment was well tolerated. Finally, alone or in association with irradiation or temozolomide, TP5 prolonged mouse survival. TP5 alone or in association with temozolomide and radiotherapy is a promising therapeutic option for glioblastoma.

scholarly journals Dominant TEL1-hy Mutations Compensate for Mec1 Lack of Functions in the DNA Damage Response

2007 ◽  
Vol 28 (1) ◽  
pp. 358-375 ◽  
Author(s):  
Veronica Baldo ◽  
Valentina Testoni ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Dna Damage ◽  
Protein Kinases ◽  
Dna Lesions ◽  
Wild Type ◽  
Checkpoint Activation ◽  
Telomere Shortening ◽  
Checkpoint Response ◽  
Double Stranded Dna ◽  
Telomere Homeostasis

ABSTRACT Eukaryotic genome integrity is safeguarded by two highly conserved protein kinases that are called ATR and ATM for humans and Mec1 and Tel1 for Saccharomyces cerevisiae. Although they share sequence similarities and substrates, these protein kinases perform different specialized functions. In particular, Mec1 plays a key role in the DNA damage checkpoint response, whereas Tel1 primarily is involved in telomere homeostasis, and its checkpoint function is masked by the prevailing activity of Mec1. In order to understand how this specificity is achieved, we searched for TEL1 mutations able to compensate for the lack of Mec1 functions. Here, we describe seven independent dominant TEL1-hy alleles that are able to suppress, to different extents, both the hypersensitivity to genotoxic agents and the checkpoint defects of Mec1-deficient cells. Most of these alleles also cause telomere overelongation. In vitro kinase activity was increased compared to that of wild-type Tel1 in the Tel1-hy385, Tel1-hy394, Tel1-hy680, and Tel1-hy909 variants, but its activity was not affected by the TEL1-hy184 and TEL1-hy628 mutations and was slightly reduced by the TEL1-hy544 mutation. Thus, the phenotypes caused by at least some Tel1-hy variants are not simply the consequence of improved catalytic activity. Further characterization shows that Tel1-hy909 not only can sense and signal a single double-stranded DNA break, unlike wild-type Tel1, but also contributes more efficiently than Tel1 to single-stranded DNA accumulation at double-strand ends, thus enhancing Mec1 signaling activity. Moreover, it causes unscheduled checkpoint activation in unperturbed conditions and upregulates the checkpoint response to small amounts of DNA lesions. Finally, Tel1-hy544 can activate the checkpoint more efficiently than wild-type Tel1, while it causes telomere shortening, indicating that the checkpoint and telomeric functions of Tel1 can be separable.

scholarly journals Nse2, a Component of the Smc5-6 Complex, Is a SUMO Ligase Required for the Response to DNA Damage

2005 ◽  
Vol 25 (1) ◽  
pp. 185-196 ◽  
Author(s):  
Emily A. Andrews ◽  
Jan Palecek ◽  
John Sergeant ◽  
Elaine Taylor ◽  
Alan R. Lehmann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ring Finger ◽  
Dependent Manner ◽  
Dna Damaging Agents ◽  
Sumo Ligase ◽  
Essential Function ◽  
Mutant Cells ◽  
Smc Proteins

ABSTRACT The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M r complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex.

scholarly journals Nucleophosmin Is Required for DNA Integrity and p19Arf Protein Stability

2005 ◽  
Vol 25 (20) ◽  
pp. 8874-8886 ◽  
Author(s):  
Emanuela Colombo ◽  
Paola Bonetti ◽  
Eros Lazzerini Denchi ◽  
Paola Martinelli ◽  
Raffaella Zamponi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Genetic Alterations ◽  
Dna Integrity ◽  
Senescent Phenotype ◽  
Tumor Suppressive Function ◽  
Proliferation In Vitro

ABSTRACT Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19Arf and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.

scholarly journals p53 Sites Acetylated In Vitro by PCAF and p300 Are Acetylated In Vivo in Response to DNA Damage

1999 ◽  
Vol 19 (2) ◽  
pp. 1202-1209 ◽  
Author(s):  
Lin Liu ◽  
Daniel M. Scolnick ◽  
Raymond C. Trievel ◽  
Hong Bing Zhang ◽  
Ronen Marmorstein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Binding Activity ◽  
Creb Binding Protein ◽  
Dna Binding Activity ◽  
Dna Damaging Agents ◽  
Lysine Residues ◽  
P53 Tumor Suppressor Protein ◽  
Histone Acetyltransferase Activity

ABSTRACT The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53’s ability to bind to its cognate DNA site. We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

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.

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