scholarly journals CDK and Mec1/Tel1-catalyzed phosphorylation of Sae2 regulate different responses to DNA damage

2019 ◽  
Vol 47 (21) ◽  
pp. 11238-11249 ◽  
Author(s):  
Tai-Yuan Yu ◽  
Valerie E Garcia ◽  
Lorraine S Symington
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Phosphorylation Site ◽  
Physiological Role ◽  
Checkpoint Signaling ◽  
Strand Breaks ◽  
Checkpoint Kinases ◽  
Damage Response ◽  
End Resection

Abstract Sae2 functions in the DNA damage response by controlling Mre11-Rad50-Xrs2 (MRX)-catalyzed end resection, an essential step for homology-dependent repair of double-strand breaks (DSBs), and by attenuating DNA damage checkpoint signaling. Phosphorylation of Sae2 by cyclin-dependent kinase (CDK1/Cdc28) activates the Mre11 endonuclease, while the physiological role of Sae2 phosphorylation by Mec1 and Tel1 checkpoint kinases is not fully understood. Here, we compare the phenotype of sae2 mutants lacking the main CDK (sae2-S267A) or Mec1 and Tel1 phosphorylation sites (sae2-5A) with sae2Δ and Mre11 nuclease defective (mre11-nd) mutants. The phosphorylation-site mutations confer DNA damage sensitivity, but not to the same extent as sae2Δ. The sae2-S267A mutation is epistatic to mre11-nd for camptothecin (CPT) sensitivity and synergizes with sgs1Δ, whereas sae2-5A synergizes with mre11-nd and exhibits epistasis with sgs1Δ. We find that attenuation of checkpoint signaling by Sae2 is mostly independent of Mre11 endonuclease activation but requires Mec1 and Tel1-dependent phosphorylation of Sae2. These results support a model whereby CDK-catalyzed phosphorylation of Sae2 activates resection via Mre11 endonuclease, whereas Sae2 phosphorylation by Mec1 and Tel1 promotes resection by the Dna2-Sgs1 and Exo1 pathways indirectly by dampening the DNA damage response.

scholarly journals Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

2018 ◽  
Vol 115 (51) ◽  
pp. E11961-E11969 ◽  
Author(s):  
Tai-Yuan Yu ◽  
Michael T. Kimble ◽  
Lorraine S. Symington
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Inhibitory Effects ◽  
Checkpoint Signaling ◽  
Synthetic Lethal ◽  
Strand Breaks ◽  
Damage Response ◽  
End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

scholarly journals Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

2018 ◽  
Author(s):  
Tai-Yuan Yu ◽  
Michael Kimble ◽  
Lorraine S Symington
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Inhibitory Effects ◽  
Checkpoint Signaling ◽  
Synthetic Lethal ◽  
Strand Breaks ◽  
Damage Response ◽  
End Resection

ABSTRACTThe Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5’-3’ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5’ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1 mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

scholarly journals SET8 Is a Potential Therapeutic Target in MM

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4435-4435
Author(s):  
Herviou Laurie ◽  
Fanny Izard ◽  
Elke De Bruyne ◽  
Eva Desmedt ◽  
Anqi Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Myeloma Cells ◽  
Strand Breaks ◽  
Damage Response

Abstract Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers. We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described. We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway. Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. Disclosures No relevant conflicts of interest to declare.

A Role for NIPA in DNA Damage Response.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1265-1265
Author(s):  
Christine von Klitzing ◽  
Florian Bassermann ◽  
Stephan W. Morris ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Phosphorylation Site ◽  
Genotoxic Stress ◽  
S Phase ◽  
Damage Response ◽  
A Cell ◽  
Cycle Dependent

Abstract The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis. Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways. In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

scholarly journals 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

2012 ◽  
Vol 197 (2) ◽  
pp. 283-300 ◽  
Author(s):  
Paula Martínez ◽  
Juana M. Flores ◽  
Maria A. Blasco
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.

scholarly journals p53 Binding Protein 53BP1 Is Required for DNA Damage Responses and Tumor Suppression in Mice

2003 ◽  
Vol 23 (7) ◽  
pp. 2556-2563 ◽  
Author(s):  
Irene M. Ward ◽  
Kay Minn ◽  
Jan van Deursen ◽  
Junjie Chen
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Tumor Suppression ◽  
Binding Protein ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Damage Response ◽  
Dna Strand ◽  
Dna Damage Response Pathway

ABSTRACT 53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM−/− mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1−/− cells show a slight S-phase checkpoint defect and prolonged G2/M arrest after treatment with ionizing radiation. Moreover, 53BP1−/− cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.

scholarly journals Regulation of the Cell-Intrinsic DNA Damage Response by the Innate Immune Machinery

2021 ◽  
Vol 22 (23) ◽  
pp. 12761
Author(s):  
Thomas J. Hayman ◽  
Peter M. Glazer
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Innate Immune ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Immune Mediated ◽  
Damage Response

Maintenance of genomic integrity is crucial for cell survival. As such, elegant DNA damage response (DDR) systems have evolved to ensure proper repair of DNA double-strand breaks (DSBs) and other lesions that threaten genomic integrity. Towards this end, most therapeutic studies have focused on understanding of the canonical DNA DSB repair pathways to enhance the efficacy of DNA-damaging therapies. While these approaches have been fruitful, there has been relatively limited success to date and potential for significant normal tissue toxicity. With the advent of novel immunotherapies, there has been interest in understanding the interactions of radiation therapy with the innate and adaptive immune responses, with the ultimate goal of enhancing treatment efficacy. While a substantial body of work has demonstrated control of the immune-mediated (extrinsic) responses to DNA-damaging therapies by several innate immune pathways (e.g., cGAS–STING and RIG-I), emerging work demonstrates an underappreciated role of the innate immune machinery in directly regulating tumor cell-intrinsic/cell-autonomous responses to DNA damage.

scholarly journals DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 504
Author(s):  
Takayuki Saitoh ◽  
Tsukasa Oda
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Tumor Microenvironment ◽  
Dna Damage Response ◽  
Genetic Instability ◽  
Dna Repair Mechanisms ◽  
Damage Response ◽  
Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

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