scholarly journals Profilin-1; a novel regulator of DNA damage response and repair machinery in keratinocytes

2021 ◽  
Author(s):  
Chang-Jin Lee ◽  
Min-Ji Yoon ◽  
Dong Hyun Kim ◽  
Tae Uk Kim ◽  
Youn-Jung Kang
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Recovery Time ◽  
Actin Polymerization ◽  
Loss Of Function ◽  
Dna Double Strand Breaks ◽  
Pten Loss ◽  
Damage Response ◽  
Specific Regulation

AbstractProfilin-1 (PFN1) regulates actin polymerization and cytoskeletal growth. Despite the essential roles of PFN1 in cell integration, its subcellular function in keratinocyte has not been elucidated yet. Here we characterize the specific regulation of PFN1 in DNA damage response and repair machinery. PFN1 depletion accelerated DNA damage-mediated apoptosis exhibiting PTEN loss of function instigated by increased phosphorylated inactivation followed by high levels of AKT activation. PFN1 changed its predominant cytoplasmic localization to the nucleus upon DNA damage and subsequently restored the cytoplasmic compartment during the recovery time. Even though γH2AX was recruited at the sites of DNA double strand breaks in response to DNA damage, PFN1-deficient cells failed to recruit DNA repair factors, whereas control cells exhibited significant increases of these genes. Additionally, PFN1 depletion resulted in disruption of PTEN-AKT cascade upon DNA damage and CHK1-mediated cell cycle arrest was not recovered even after the recovery time exhibiting γH2AX accumulation. This might suggest PFN1 roles in regulating DNA damage response and repair machinery to protect cells from DNA damage. Future studies addressing the crosstalk and regulation of PTEN-related DNA damage sensing and repair pathway choice by PFN1 may further aid to identify new mechanistic insights for various DNA repair disorders.

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.

scholarly journals POGZ modulates the DNA damage response in a HP1-dependent manner

2021 ◽  
Author(s):  
John Heath ◽  
Estelle Simo Cheyou ◽  
Steven Findlay ◽  
Vincent Luo ◽  
Edgar Pinedo Carpio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Genome Stability ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Humoral Immune ◽  
Damage Response

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonic lethal and Pogz haplo-insufficiency (Pogz+/Δ) results in a developmental delay, a deficit in intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/Δ mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

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.

Increased γH2AX Foci in Old Hematopoietic Stem Cells Are Independent of the DNA Damage Response and Linked to Inefficient DNA Replication/Transcription

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1207-1207
Author(s):  
Johanna Flach ◽  
Sietske Bakker ◽  
Pauline Conroy ◽  
Damien Reynaud ◽  
Michelle M. Le Beau ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Stem ◽  
Strand Breaks ◽  
Γh2ax Foci ◽  
Damage Response

Abstract Abstract 1207 Hematopoietic stem cells (HSCs) are responsible for the life-long production of all blood cells. Changes in the biological function of old HSCs have been directly linked to the occurrence of age-related blood defects including immunosenescence, anemia and the development of a broad spectrum of hematological disorders (i.e., myeloproliferative neoplasms, leukemia, bone marrow failure). Gene expression studies and analysis of genetically modified mice have also suggested that error-prone DNA repair as well as a decrease in genomic stability are one of the driving forces for the reduced functional capacity of old HSCs. Here, we used HSCs (Lin-/c-Kit+/Sca-1+/Flk2-/CD48-/CD150+) isolated from the bone marrow of old (20–24 months old) and young (6–12 weeks old) C57Bl/6 mice to directly investigate the DNA damage response of old HSCs. Using immunofluorescence, we first confirmed that freshly isolated quiescent old HSCs have an increased number of γH2AX foci, which is a well-established indicator for DNA double-strand breaks. In addition, we found that these intrinsically occurring γH2AX foci specifically co-localized with nucleolar markers (i.e., UBF, fibrillarin and nucleolin) and were a cell-intrinsic feature of old HSCs as demonstrated by transplantation experiments of old HSCs into young recipient mice. However, we could not demonstrate that nucleolar γH2AX foci in old HSCs represent sites of DNA damage. Neither did known DNA repair-associated markers like 53BP1 co-localize with nucleolar γH2AX foci, nor were other DNA damage markers such as phospho-ATM or PARP1 increased in old HSCs. In addition, none of the other methods we used to measure DNA fragmentation such as TUNEL or COMET assays revealed elevated levels of DNA damage in old HSCs, and spectral karyotyping (SKY) analysis of in vitro cultured old HSCs did not provide evidence for DNA damage-associated chromosomal alterations. We then used 2Gy ionizing radiation (IR) to directly induce DNA double-strand breaks and measured the DNA repair capacity of old HSCs. Strikingly, we observed a similar DNA damage response and DNA repair kinetics in young and old HSCs. These results provide evidence that old HSCs can respond adequately to DNA damage and that accumulation of γH2AX at the nucleolus is not the consequence of an activated DNA damage response. The nucleolus consists of a highly regulated repetitive sequence of rDNA units and is the site of ribosomal DNA transcription. Both young and old quiescent HSCs have well-formed nucleoli as shown by electron microscopy analyses. Strikingly, we found a complete disappearance of nucleolar γH2AX foci when old HSCs are forced into cell cycle upon in vitro culture. Furthermore, we observed a significant delay in the onset of the first cell division and timing of nucleolar reformation following mitosis in old HSCs. In addition, cycling old HSCs displayed higher levels of DNA replication/transcription-associated γH2AX foci compared to cycling young HSCs. We are currently investigating how defects in the DNA replication machinery could contribute to the nucleolar γH2AX foci and cell cycle features of old HSCs, and whether old HSCs maintain similar levels of rRNA transcription as compared to young HSCs. Taken together, our results demonstrate that the increased numbers of γH2AX foci observed in old HSCs is not caused by an accumulation of DNA damage. Instead, nucleolar γH2AX foci appear to be a hallmark feature of quiescent old HSCs that could reflect epigenetic changes in rDNA chromatin structure linked to inefficient DNA replication/transcription. Disclosures: No relevant conflicts of interest to declare.

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 DROSHA associates to DNA damage sites and is required for DNA repair

2018 ◽  
Author(s):  
Matteo Cabrini ◽  
Marco Roncador ◽  
Alessandro Galbiati ◽  
Lina Cipolla ◽  
Fabio Iannelli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Dna Lesions ◽  
Cellular System ◽  
Proximity Ligation Assay ◽  
Dna Double Strand Breaks ◽  
Proximity Ligation ◽  
Damage Response ◽  
Non Homologous End Joining

AbstractThe DNA damage response (DDR) is the signaling cascade through which a cell recognizes DNA lesions, and promotes their resolution via the repair pathways of Non-Homologous End Joining (NHEJ), or Homologous Recombination (HR). We recently demonstrated that DROSHA boosts DDR signaling by processing damage-induced long non-coding RNAs into smaller DNA damage response RNAs (DDRNAs). However, the location at which DROSHA exerts its DDR functions, relative to sites of DNA damage, remains unknown.To investigate DROSHA’s localization during DDR activation, we used the DiVA cellular system, which allows the controlled induction of several DNA double strand breaks (DSBs) in the human genome. Indeed, by genome wide chromatin immunoprecipitation followed by next generation sequencing, we demonstrate that DROSHA associates with DSBs. In support of this, DSB-recruitment of DROSHA is detectable at the single-cell level by Proximity Ligation Assay between DROSHA and known DDR markers, and by DNA damage in situ ligation followed by Proximity Ligation Assay (DI-PLA), which demonstrates proximity of DROSHA to DNA ends. DROSHA recruitment occurs at both genic and inter-genic DSBs, suggesting that its recruitment is independent from ongoing transcription preceding damage generation. DROSHA’s recruitment to DNA lesions occurs throughout the cell cycle, and with a preference for NHEJ-prone DSBs. Consistently, inhibition of the HR pathway increases DROSHA recruitment, and DROSHA knock down strongly impairs NHEJ efficiency in a GFP-reporter cellular system for monitoring NHEJ DNA repair. Overall, these results demonstrate that DROSHA acts locally at sites of DNA damage to promote NHEJ DNA repair.

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.

scholarly journals Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1156
Author(s):  
Jiachen Xuan ◽  
Kezia Gitareja ◽  
Natalie Brajanovski ◽  
Elaine Sanij
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Therapy ◽  
Dna Damage Response ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Pol I ◽  
Clinical Investigations ◽  
Damage Response ◽  
Synthesis And Processing

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.

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