Caspase-2 cleaves DNA fragmentation factor (DFF45)/inhibitor of caspase-activated DNase (ICAD)

To investigate the signal transduction pathway of caspase-2, cell permeable Tat-reverse-caspase-2 was constructed, characterized and utilized for biochemical and cellular studies. It could induce the cell death as early as 2 h, and caspase-2-specific VDVADase activity but not other caspase activities including DEVDase and IETDase. Interestingly, nuclear DNA fragmentation occurred and consistently DNA fragmentation factor (DFF45)/Inhibitor of caspase-activated DNase (ICAD) was cleaved inside the cell as well as in vitro, suggesting a role of caspase-2 in nuclear DNA fragmentation.     

Uhrf2 is important for DNA damage response in vascular smooth muscle cells

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.     

Condensin recruitment to chromatin is inhibited by Chk2 kinase in response to DNA damage

The DNA damage checkpoint, when activated in response to genotoxic damage during S phase, arrests cells in G2 phase of the cell cycle. ATM, ATR, Chk1 and Chk2 kinases are the main effectors of this checkpoint pathway. The checkpoint kinases prevent the onset of mitosis by eliciting well characterized inhibitory phosphorylation of Cdk1. Since Cdk1 is required for the recruitment of condensin, it is thought that upon DNA damage the checkpoint also indirectly blocks chromosome condensation via Cdk1 inhibition. Here we report that the G2 damage checkpoint prevents stable recruitment of the chromosome-packaging-machinery components condensin complex I and II onto the chromatin even in the presence of an active Cdk1. DNA damage-induced inhibition of condensin subunit recruitment is mediated specifically by the Chk2 kinase, implying that the condensin complexes are targeted by the checkpoint in response to DNA damage, independently of Cdk1 inactivation. Thus, the G2 checkpoint directly prevents stable recruitment of condensin complexes to actively prevent chromosome compaction during G2 arrest, presumably to ensure efficient repair of the genomic damage.     

Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53^−/− mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.     

NFBD1/MDC1 stabilizes oncogenic MDM2 to contribute to cell fate determination in response to DNA damage

In response to DNA damage, NFBD1/MDC1 induces the accumulation of DNA repair machinery such as MRN complex at the sites of damaged DNA to form nuclear foci. In this study, we found that NFBD1 directly interacts with MDM2 and increases its stability. During adriamycin (ADR)-mediated apoptosis, expression levels of NFBD1 reduced in association with the down-regulation of MDM2. Enforced expression of NFBD1 resulted in a significant stabilization of MDM2. Consistent with these observations, siRNA-mediated knockdown of the endogenous NFBD1 decreased the amounts of the endogenous MDM2. Immunoprecipitation and in vitro pull-down assays demonstrated that NFBD1 interacts with MDM2 through its COOH-terminal BRCT domains. In accordance with our recent results, enforced expression of NFBD1 rendered cells resistant to DNA damage. Similar results were also obtained in cells expressing exogenous MDM2. Taken together, our present findings suggest that NFBD1-mediated stabilization contributes to cell survival in response to DNA damage.     

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]     

Spatially distinct functions of Clb2 in the DNA damage response

In budding yeast four mitotic cyclins (Clb1–4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G₂/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.     

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

We previously reported that TrkA overexpression causes accumulation of γH2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we established TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and γH2AX in TrkA-induced cell death. γH2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.     

Molecular interaction map of the p53 and Mdm2 logic elements, which control the Off-On switch of p53 in response to DNA damage

The molecular network that controls responses to genotoxic stress is centered at p53 and Mdm2. Recent findings have shown this network to be more complex than previously envisioned. Using a notation specifically designed for circuit diagram-like representations of bioregulatory networks, we have prepared an updated molecular interaction map of the immediate connections of p53 and Mdm2, which are described as logic elements of the network. We use the map as the basis for a comprehensive review of current concepts of signal processing by these logic elements (an interactive version of the maps-eMIMs can be examined at ). We also used molecular interaction maps to propose a p53 Off-On switch in response to DNA damage.     

CHK2 kinase in the DNA damage response and beyond

The serine/threonine kinase CHK2 is a keycomponent of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates 〉20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In par- ticular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.     

Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia

Caspase-3 has been identified as a key protease that, by targeting a limited number of proteins, can disrupt essential homeostatic processes and initiate an orderly disassembly of cells, including degradation of genomic DNA. We demonstrate the usefulness of an antibody specific for activated caspase-3 in a model of neonatal rat hypoxia-ischemia (Hl) and correlate the spatial and temporal activation of caspase-3 with three different markers of DNA damage and with the loss of a neuronal marker [microtubule-associated protein 2 (MAP 2)]. An oligonucleotide hairpin probe (HPP) with one base overhang in the 3' end displayed a close colocalization with caspase-3 activation at 3 h post-Hl, whereas terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) appeared later (24 h post-Hl). A monoclonal antibody against single-stranded DNA appeared to stain an entirely different population of cells, not positive for active caspase-3, HPP, or TUNEL at this time point. After 24 h of reperfusion, however, when cellular injury is extensive, all markers stained a large number of cells with a high degree of colocalization, and all markers delineated regions with loss of MAP 2. We conclude that the HPP shows the best correlation with pathological caspase-3 activation in this model.     

Studying the DNA damage response using in vitro model systems

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order to prevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathways that delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis of ATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro model systems to understand the detailed mechanisms underlying their function. Here we describe some recent findings obtained analysing these pathways using in vitro model systems. In particular we focus on the studies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response in the context of the cell cycle.     

Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay

A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.     

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.     

Chromatin modulation and the DNA damage response

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.     

DNA damage in response to an Ironman triathlon

The major aims of this study were to investigate the effect of an Ironman triathlon on DNA migration in the single cell gel electrophoresis assay, apoptosis and necrosis in the cytokinesis-block micronucleus cytome assay with lymphocytes and on changes of total antioxidant capacity in plasma. Blood samples were taken 2 days (d) before, within 20 min, 1 d, 5 d and 19 d post-race. The level of strand breaks decreased (p<0.05) immediately after the race, then increased (p<0.01) 1 d post-race and declined (p<0.01) until 19 d post-race. Apoptotic and necrotic cells decreased (p<0.01) and the total antioxidant status increased (p<0.01) immediately after the race. The results indicate that ultra-endurance exercise does not cause prolonged DNA damage in well-trained male athletes.     

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.