Autophagy acts as a safeguard mechanism against G-quadruplex ligand-mediated DNA damage

G-quadruplex ligands have attracted considerable interest as novel anticancer therapeutics due to their capability to interfere with guanosine-rich DNA/RNA sequences, such as telomeres. Elucidation of the structures of telomeric G-quadruplexes has led, in the past few years, to the rational development of effective G-quadruplex-stabilizing small molecules. In the present study, we showed that short-term exposure of melanoma cells to Ant1,5—an anthracene-based ligand able to stabilize telomeric G-quadruplexes—impaired cell growth without inducing cell senescence or apoptosis. Conversely, drug-treated cells were characterized by the occurrence of typical biochemical and morphological features associated with autophagy, such as an increase in the lipidated form of the autophagic marker LC3B and the accumulation of autophagosomes. Such drug-induced autophagy occurred as a consequence of DNA damage induction, at least in part dependent on drug-mediated telomere uncapping, through a pathway converging on the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Indeed, melanoma cells depleted for CDKN1A did not show evidence of autophagic markers upon exposure to Ant1,5. The inhibition of autophagy by a pharmacologic inhibitor or through RNAi-mediated depletion of the ATG5 gene enhanced the cytotoxic activity of Ant1,5, as revealed by the marked increase in drug-induced apoptosis. Our data outline a molecular scenario in which G-quadruplex ligand-induced telomeric dysfunctions and DNA damage are translated into an autophagic response and provide the first evidence of autophagy as a safeguard mechanism activated by melanoma cells to counteract G-quadruplex ligand-mediated cellular stress.     

Polo-like kinase-1 in DNA damage response

Polo-like kinase-1 (Plk1) belongs to a family of serine-threonine kinases and plays a critical role in mitotic progression. Plk1 involves in the initiation of mitosis, centrosome maturation, bipolar spindle formation, and cytokinesis, well-reported as traditional functions of Plk1. In this review, we discuss the role of Plk1 during DNA damage response beyond the functions in mitotsis. When DNA is damaged in cells under various stress conditions, the checkpoint mechanism is activated to allow cells to have enough time for repair. When damage is repaired, cells progress continuously their division, which is called checkpoint recovery. If damage is too severe to repair, cells undergo apoptotic pathway. If damage is not completely repaired, cells undergo a process called checkpoint adaptation, and resume cell division cycle with damaged DNA. Plk1 targets and regulates many key factors in the process of damage response, and we deal with these subjects in this review. [BMB Reports 2014; 47(5): 249-255]     

Site-1 protease inhibits mitochondrial respiration by controlling the TGF-β target gene Mss51

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S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and γH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.     

HIGD1A links SIRT1 activity to adipose browning by inhibiting the ROS/DNA damage pathway

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A distinct response to endogenous DNA damage in the development of Nbs1-deficient cortical neurons

Microcephaly is a clinical characteristic for human nijmegen breakage syndrome (NBS, mutated in NBS1 gene), a chromosomal instability syndrome. However, the underlying molecular pathogenesis remains elusive. In the present study, we demonstrate that neuronal disruption of NBS (Nbn in mice) causes microcephaly characterized by the reduction of cerebral cortex and corpus callosum, recapitulating neuronal anomalies in human NBS. Nbs1-deficient neocortex shows accumulative endogenous DNA damage and defective activation of Ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway upon DNA damage. Notably, in contrast to massive apoptotic cell death in Nbs1-deficient cerebella, activation of p53 leads to a defective neuroprogenitor proliferation in neocortex, likely via specific persistent induction of hematopoietic zinc finger (Hzf) that preferentially promotes p53-mediated cell cycle arrest whilst inhibiting apoptosis. Moreover, Trp53 mutations substantially rescue the microcephaly in Nbs1-deficient mice. Thus, the present results reveal the first clue that developing neurons at different regions of brain selectively respond to endogenous DNA damage, and underscore an important role for Nbs1 in neurogenesis.     

mTORC1 pathway in DNA damage response

Living organisms have evolved various mechanisms to control their metabolism and response to various stresses, allowing them to survive and grow in different environments. In eukaryotes, the highly conserved mechanistic target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cellular metabolism, proliferation and survival. A growing body of evidence indicates that mTOR signaling is closely related to another cellular protection mechanism, the DNA damage response (DDR). Many factors important for the DDR are also involved in the mTOR pathway. In this review, we discuss how these two pathways communicate to ensure an efficient protection of the cell against metabolic and genotoxic stresses. We also describe how anticancer therapies benefit from simultaneous targeting of the DDR and mTOR pathways.     

Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.     

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double‐strand breaks (DSBs), but only in the G1‐phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1–2 and 7–8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1–2 and 4–5. The BRCT domains 4–5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S‐phase, we observed a checkpoint defect after siRNA‐mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.     

53BP1与DNA双链断裂损伤修复

DNA的精确复制和遗传对维持基因组稳定性有重要作用。DNA双链断裂损伤可能诱导细胞凋亡和染色质重排,在肿瘤的发生发展过程中发挥作用。53BP1是DNA双链断裂修复中的重要调节蛋白质之一,对调控损伤修复平衡和维持基因组稳定性起着重要作用。本文主要对53BP1的结构、生物学功能、信号通路、分子机制和翻译后修饰做一浅显的总结和展望,希望能为53BP1的深入研究提供一些理论基础。     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Nbs1‐mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis

ObjectivesDNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood‐derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well‐defined.Materials and methodsMice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1‐p53 signaling in HSCs and haematopoietic progenitors.ResultsNbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm‐Chk2‐p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality.ConclusionsOur study discloses that DNA double‐strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.     

A comparison of BRCA1 nuclear localization with 14 DNA damage response proteins and domains: Identification of specific differences between BRCA1 and 53BP1 at DNA damage-induced foci

BRCA1 is an important mediator of the DNA damage response pathway. Previous studies have identified a number of proteins that associate with BRCA1 at nuclear foci after ionizing radiation (IR)-induced DNA damage. However, the co-localization patterns of BRCA1 and various DNA damage response proteins have not yet been systematically quantified and compared within the same experimental system. In this study, a new inducible human cell line was established to allow unambiguous detection of YFP–BRCA1 at nuclear foci. Quantitative 2-D microscopic analysis was performed to compare the intranuclear co-localization of YFP–BRCA1 with 10 cellular proteins and 4 cellular domains before and after IR. Intriguingly, YFP–BRCA1 displayed significantly better focal co-localization with BARD1, RAP80 and Abraxas than with the upstream foci-initiating proteins γH2AX and MDC1. In contrast to previous reports, we found that the co-localization between YFP–BRCA1 and 53BP1 foci was surprisingly weak. Quantitative analyses of 3-D confocal images showed that ~ 60% of 53BP1 foci were unrelated to YFP–BRCA1 foci, ~ 35% of foci were abutting and only ~ 5% of foci co-localized. The YFP–BRCA1 and 53BP1 nuclear foci were distinctively separated within the first 3 h after IR. In addition, in situ nuclear retention analysis revealed YFP–BRCA1 and BARD1 are less mobile than 53BP1 at IR-induced nuclear foci. Our findings indicate that BRCA1–BARD1 and 53BP1 are proximal but not overlapping at DNA break sites and are consistent with recent evidence for distinct roles of these proteins in the DNA damage response pathway.     

Palmdelphin,a novel target of p53 with Ser46 phosphorylation,controls cell death in response to DNA damage

The tumor suppressor gene p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends on mainly its phosphorylation. Particularly, the phosphorylation at serine-46 of p53 is indispensable in promoting pro-apoptotic genes that are, however, poorly determined. In the current study, we identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated serine-46-specific manner. Upregulation of palmdelphin was observed in wild-type p53-transfected cells, but not in serine-46-mutated cells. Expression of palmdelphin was induced by p53 in response to DNA damage. In turn, palmdelphin induced apoptosis. Intriguingly, downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion. Upon DNA damage, palmdelphin dominantly accumulated in the nucleus to induce apoptosis. These findings define palmdelphin as a target of serine-46-phosphorylated p53 that controls cell death in response to DNA damage.     

DNA damage induced activation of Cygb stabilizes p53 and mediates G1 arrest

Cytoglobin (Cygb) is an emerging tumor suppressor gene silenced by promoter hypermethylation in many human tumors. So far, the precise molecular mechanism underlying its tumor suppressive function remains poorly understood. Here, we identified Cygb as a genotoxic stress-responsive hemoprotein upregulated upon sensing cellular DNA damage. Our studies demonstrated that Cygb physically associates with and stabilizes p53, a key cellular DNA damage signaling factor. We provide evidence that Cygb extends the half-life of p53 by blocking its ubiquitination and subsequent degradation. We show that, upon DNA damage, cells overexpressing Cygb displayed proliferation defect by rapid accumulation of p53 and its target gene p21, while Cygb knockdown cells failed to efficiently arrest in G1 phase in response to DNA insult. These results suggest a possible involvement of Cygb in mediating cellular response to DNA damage and thereby contributing in the maintenance of genomic integrity. Our study thus presents a novel insight into the mechanistic role of Cygb in tumor suppression.     

16p11.2 haploinsufficiency reduces mitochondrial biogenesis in brain endothelial cells and alters brain metabolism in adult mice

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DNA‐PK suppresses a p53‐independent apoptotic response to DNA damage

p53 is required for DNA damage‐induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53‐deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA‐dependent protein kinase (DNA‐PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation‐induced apoptosis, whereas apoptosis in DNA‐PK_cs/p53, Ku80/p53 and Ku70/p53 double‐null mice was quantitatively equivalent to that seen in wild‐type mice. This p53‐independent apoptotic response was specific to the loss of DNA‐PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double‐null mice. Furthermore, it was associated with an increase in phospho‐checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA‐PK, does not require p53.