scholarly journals Recent advances in the nucleolar responses to DNA double-strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9449-9461
Author(s):  
Lea Milling Korsholm ◽  
Zita Gál ◽  
Blanca Nieto ◽  
Oliver Quevedo ◽  
Stavroula Boukoura ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Ribosomal Dna ◽  
Repetitive Sequences ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

Synthetic Lethality In CLL With DNA Damage Response Defect By Targeting ATR Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 120-120
Author(s):  
Tatjana Stankovic ◽  
Davies Nicholas ◽  
Marwan Kwok ◽  
Edward Smith ◽  
Eliot Yates ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Synthetic Lethality ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Single Strand Break ◽  
Strand Breaks ◽  
Damage Response ◽  
Repair Pathways

Abstract Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed. ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality’ and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM. First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea. We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support. To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss. We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures: Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

scholarly journals The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

2010 ◽  
Vol 191 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Ye Xu ◽  
Yingli Sun ◽  
Xiaofeng Jiang ◽  
Marina K. Ayrapetov ◽  
Patryk Moskwa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Active Process ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Significant Barrier ◽  
Nucleosome Stability

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.

High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair

2003 ◽  
Vol 285 (2) ◽  
pp. F266-F274 ◽  
Author(s):  
Natalia I. Dmitrieva ◽  
Dmitry V. Bulavin ◽  
Maurice B. Burg
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Damage Response

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.

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.

Heterochromatin and the DNA damage response: the need to relaxThis paper is one of a selection of papers in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (1) ◽  
pp. 45-60 ◽  
Author(s):  
Kendra L. Cann ◽  
Graham Dellaire
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Chromatin Structure ◽  
Peer Review Process ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Damage Response ◽  
Chromosomal Mutations

Higher order chromatin structure has an impact on all nuclear functions, including the DNA damage response. Over the past several years, it has become increasingly clear that heterochromatin and euchromatin represent separate entities with respect to both damage sensitivity and repair. The chromatin compaction present in heterochromatin helps to protect this DNA from damage; however, when lesions do occur, the compaction restricts the ability of DNA damage response proteins to access the site, as evidenced by its ability to block the expansion of H2AX phosphorylation. As such, DNA damage in heterochromatin is refractory to repair, which requires the surrounding chromatin structure to be decondensed. In the case of DNA double-strand breaks, this relaxation is at least partially mediated by the ATM kinase phosphorylating and inhibiting the function of the transcriptional repressor KAP1. This review will focus on the functions of KAP1 and other proteins involved in the maintenance or restriction of heterochromatin, including HP1 and TIP60, in the DNA damage response. As heterochromatin is important for maintaining genomic stability, cells must maintain a delicate balance between allowing repair factors access to these regions and ensuring that these regions retain their organization to prevent increased DNA damage and chromosomal mutations.

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.

scholarly journals Screen identifies DYRK1B network as mediator of transcription repression on damaged chromatin

2020 ◽  
Vol 117 (29) ◽  
pp. 17019-17030 ◽  
Author(s):  
Chao Dong ◽  
Kirk L. West ◽  
Xin Yi Tan ◽  
Junshi Li ◽  
Toyotaka Ishibashi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Gene Silencing ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Active Chromatin ◽  
Chemical Library ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Damage Response

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B–EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

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