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The DNA‐dependent protein kinase (DNA‐PK) is an abundant nuclear protein that mediates DNA double‐strand break repair by nonhomologous end joining (NHEJ). As such, DNA‐PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA‐PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high‐performance liquid chromatography‐mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid–liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4 μm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive‐mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1–300 ng/mL for the metabolite and proved to be both accurate (97.3%–103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography‐tandem mass spectroscopy (LC–MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics. [ABSTRACT FROM AUTHOR]

Background: Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE‐13) and to investigate their underlying mechanisms. Methods: Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA‐repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double‐strand break (DSB) repair were measured by western blot. Results: Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation‐induced inhibition of clonogenic survival in ECA109 and TE‐13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE‐13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. Conclusions: These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC. [ABSTRACT FROM AUTHOR]

The cyclic GMP‐AMP synthase–stimulator of interferon genes (cGAS‐STING) pathway is central for the initiation of anti‐tumoural immune responses. Enormous effort has been made to optimise the design and administration of STING agonists to stimulate tumour immunogenicity. However, in certain contexts the cGAS‐STING axis fuels tumourigenesis. Here, we review recent findings on the regulation of cGAS expression and activity. We particularly focus our attention on the DNA‐dependent protein kinase (DNA‐PK) complex, that recently emerged as an activator of inflammatory responses in tumour cells. We propose that stratification analyses on cGAS and DNA‐PK expression/activation status should be carried out to predict treatment efficacy. We herein also provide insights into non‐canonical functions borne by cGAS and cGAMP, highlighting how they may influence tumourigenesis. All these parameters should be taken into consideration concertedly to choose strategies aiming to effectively boost tumour immunogenicity. [ABSTRACT FROM AUTHOR]

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP‐AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro‐inflammatory signaling. We show that the DNA‐PK DNA repair complex (i) drives cGAS‐independent IRF3‐mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS‐dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA‐PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS‐dependent signaling is acquired during tumorigenesis and that cGAS and DNA‐PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity. Synopsis: Upon cytosolic DNA detection, the DNA damage repair kinase DNA‐PK promotes optimal type I Interferon and chemokine production by regulating IRF3 activation and enabling cGAS‐dependent cGAMP production. This cooperation fuels cancer‐mediated inflammation, affecting both tumour initiation and patient prognosis.DNA‐PK promotes STING‐independent IRF3‐dependent type I Interferon responses.DNA‐PK enhances genotoxic stress‐dependent type I Interferon signaling in the absence of cGAS.DNA‐PK and cGAS cooperate for optimal type I Interferon responses and chemokine/cytokine production.DNA‐PK enables cGAS‐dependent cGAMP production.cGAS expression compromises early tumorigenesis but promotes cancer‐associated chronic inflammation. [ABSTRACT FROM AUTHOR]

Background: DNA‐dependent protein kinase (DNA‐PK) is engaged in DNA damage repair and is significantly expressed in triple negative breast cancer (TNBC). Inhibiting DNA‐PK to reduce DNA damage repair provides a possibility of tumor treatment. NU7441, a DNA‐PK inhibitor, can regulate the function and differentiation of CD4+T cells and effectively enhance immunogenicity of monocyte‐derived dendritic cells. However, the effect of NU7441 on the tumor progression activity of immunosuppressive myeloid‐derived suppressor cells (MDSCs) in TNBC remains unclear. Results: In this study, we found that NU7441 alone significantly increased tumor growth in 4 T1 (a mouse TNBC cell line) tumor‐bearing mice. Bioinformatics analysis showed that DNA‐PK and functional markers of MDSCs (iNOS, Arg1, and IDO) tended to coexist in breast cancer patients. The mutations of these genes were significantly correlated with lower survival in breast cancer patients. Moreover, NU7441 significantly decreased the percentage of MDSCs in peripheral blood mononuclear cells (PBMCs), spleen and tumor, but enhanced the immunosuppressive function of splenic MDSCs. Furthermore, NU7441 increased MDSCs' DNA‐PK and pDNA‐PK protein levels in PBMCs and in the spleen and increased DNA‐PK mRNA expression and expression of MDSCs functional markers in splenic MDSCs from tumor‐bearing mice. NU7441 combined with gemcitabine reduced tumor volume, which may be because gemcitabine eliminated the remaining MDSCs with enhanced immunosuppressive ability. Conclusions: These findings highlight that the regulation of DNA‐PK activity by NU7441 promotes TNBC progression via enhancing the immunosuppressive function of MDSCs. Moreover, NU7441 combined with gemcitabine offers an efficient therapeutic approach for TNBC and merits deeper investigation. [ABSTRACT FROM AUTHOR]

DNA double‐strand breaks (DSBs) are one of the most serious types of DNA damage. However, multiple repair pathways are present in cells to ensure rapid and appropriate repair of DSBs. Pathway selection depends on several factors including cell type, cell cycle phase, and damage severity. Ribosomal protein S3 (rpS3), a component of the 40S small ribosomal subunit, is a multi‐functional protein primarily involved in protein synthesis. rpS3 is also involved in the mediation of various extra‐ribosomal pathways, including DNA damage processing and the stress response. Here, we report that rpS3 is a novel negative regulator of non‐homologous end joining (NHEJ)‐mediated repair of DSBs. We found that rpS3 interacts with the Ku heterodimers of the DNA‐dependent protein kinase (DNA‐PK) complex and slows down NHEJ ligation reactions, ultimately triggering p53‐dependent cell death following treatment with high‐dose ionizing radiation. After DSB formation, DNA‐PK phosphorylates rpS3, which consequently reduces the binding of rpS3 to the Ku complex. We hypothesized that rpS3 may play a role in DSB repair by repressing NHEJ, while inducing other repair pathways, and by initiating DSB‐induced cell death in response to severe DNA damage. [ABSTRACT FROM AUTHOR]

It has been reported that Ras‐ERK signaling regulated tumor suppressive genes via epigenetic mechanisms. Herein, we set out to investigate the correlation between K‐Ras‐ERK1/2 signaling and H1.2 phosphorylation, to provide a better understanding of K‐Ras‐ERK signaling in cancer. A plasmid for expression of mutated K‐Ras was transfected into human bladder carcinoma HT1197 cells. Western blot was carried out for testing the expression changes of ERK1/2 and H1.2. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay, soft‐agar colony formation assay, and transwell assay were used to test the effects of H1.2 phosphorylation at T146 (H1.2 T146ph) on HT1197 cells growth and migration. Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and chromatin immunoprecipitation (ChIP) were performed to test whether H1.2 T146ph regulated K‐Ras‐ERK1/2 downstream genes. Furthermore, how K‐Ras‐ERK1/2 regulated H1.2 T146ph expression was studied. We found that the ERK1/2 was activated when K‐Ras was mutated, and H1.2 T146ph expression was significantly downregulated by K‐Ras mutation. H1.2 T146E for mimicking H1.2 T146ph significantly attenuated K‐Ras mutation induced increases in HT1197 cells viability, colony formation, and relative migration. Besides, H1.2 T146ph regulated the transcription of K‐Ras‐ERK1/2 downstream genes, including NT5E, GDF15, CARD16, CYR61, IGFBP3, and WNT16B. Furthermore, K‐Ras‐ERK1/2 signaling inhibited H1.2 phosphorylation at T146 through degradation of DNA‐PK, and the degraded DNA‐PK by K‐Ras‐ERK1/2 possibly via modulation of MDM2. In conclusion, the activation of K‐Ras‐ERK1/2 signaling will repress the phosphorylation of H1.2 at T146, and thereby, promoted the growth and migration of bladder cancer cells. K‐Ras‐ERK1/2 signaling repressed H1.2 phosphorylation possibly by MDM2‐mediated degradation of DNA‐PK. HIGHLIGHTS: 1.H1.2 phosphorylation at T146 is specifically regulated by Ras‐ERK1/2 signaling. 2.H1.2T146ph inhibits the growth and migratory capacity of HT1197 cells. 3.H1.2T146ph regulates the transcription of Ras‐ERK1/2 downstream genes. 4.Ras‐ERK1/2 signaling regulates H1.2 T146ph via DNA‐PK. 5.Ras‐ERK1/2 signaling degrades DNA‐PK via MDM2. [ABSTRACT FROM AUTHOR]

Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (~200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of *#126;20 nm to analyze the focal nanostructure of the DSB marker histone γH2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained γH2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that γH2AX foci consisted of distinct circular subunits ("nanofoci") with a diameter of *#126;45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that γH2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual γH2AX-containing nucleosomes. dSTORM-based γH2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of γH2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between *#126;0.6 and *#126;1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells. [ABSTRACT FROM AUTHOR]

The Ets‐1 oncoprotein is a transcription factor that promotes target gene expression in specific biological processes. Typically, Ets‐1 activity is low in healthy cells, but elevated levels of expression have been found in cancerous cells, specifically related to tumor progression. Like the vast majority of the cellular effectors, Ets‐1 does not act alone but in association with partners. Given the important role that is attributed to Ets‐1 in major human diseases, it is crucial to identify its partners and characterize their interactions. In this context, two DNA‐repair enzymes, PARP‐1 and DNA‐PK, have been identified recently as interaction partners of Ets‐1. We here identify their binding mode by means of protein docking. The results identify the interacting surface between Ets‐1 and the two DNA‐repair enzymes centered on the α‐helix H1 of the ETS domain, leaving α‐helix H3 available to bind DNA. The models highlight a hydrophobic patch on Ets‐1 at the center of the interaction interface that includes three tryptophans (Trp338, Trp356, and Trp361). We rationalize the binding mode using a series of computational analyses, including alanine scanning, molecular dynamics simulation, and residue centrality analysis. Our study constitutes a first but important step in the characterization, at the molecular level, of the interaction between an oncoprotein and DNA‐repair enzymes. [ABSTRACT FROM AUTHOR]

DNA‐dependent protein kinase (DNA‐PK) is a very large holoenzyme comprised of the p470 kDa DNA‐PK catalytic subunit (DNA‐PKcs) and the Ku heterodimer consisting of the p86 (Ku 80) and p70 (Ku 70) subunits. It is best known for its nonhomologous end joining (NHEJ) activity, which repairs double‐strand DNA (dsDNA) breaks (DSBs). As expected, the absence of DNA‐PK activity results in sensitivity to ionizing radiation, which generates DSBs and defect in lymphocyte development, which requires NHEJ of the V(D)J region in the immunoglobulin and T‐cell receptor loci. DNA‐PK also has been reported to have functions seemingly unrelated to NHEJ. For example, DNA‐PK responds to insulin signaling to facilitate the conversion of carbohydrates to fatty acids in the liver. More recent evidence indicates that DNA‐PK activity increases with age in skeletal muscle, promoting mitochondrial loss and weight gain. These discoveries suggest that our understanding of DNA‐PK is far from complete. As many excellent reviews have already been written about the role of DNA‐PK in NHEJ, here we will review the non‐NHEJ role of DNA‐PK with a focus on its role in aging and energy metabolism. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are less frequent than single-strand breaks but have more harmful consequences on cell survival and physiology. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two main pathways that are responsible for DSB repair in eukaryotic cells, but their importance for the preservation of genome stability in totipotent blastomeres of early developing embryos has not been determined. In this study, we observed that the chemical inhibition of HR or both pathways, but not NHEJ alone, increased the number of DSBs, reduced embryo development to the blastocyst stage, and resulted in embryos with higher proportions of apoptotic cells. Targeted knockdown of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related; HR regulators) and DNA-dependent protein kinase (NHEJ regulator) mRNAs revealed that the attenuation of HR or both HR and NHEJ regulators severely impaired blastocyst formation and quality. Attenuation of ATM alone resulted in a higher incidence of DSBs, lower development and embryo quality, and increased mRNA abundance of genes that are involved in either repair pathway. These findings indicate that HR is the main pathway responsible for the promotion of DSB repair in early developing embryos, and that ATM seems to be more important than ATR in the regulation of the HR pathway in mammalian embryos. [ABSTRACT FROM AUTHOR]

Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation. DNA-damage associated with differentiation is accompanied by phosphorylation of Histone 2 AX, an event normally catalysed by kinases ATR, ATM or DNA- PK. However, the kinase responsible for phosphorylation during differentiation is not known. Here we show that inhibition of DNA- PK, but not of ATR or ATM, blocked histone phosphorylation during differentiation. We also show that DNA- PK inhibition and si RNA-mediated DNA- PK knockdown blocked cell fusion. These data implicate a new role for DNA- PK in myogenic differentiation. [ABSTRACT FROM AUTHOR]

BRCA1, a key factor in homologous recombination (HR) repair may also regulate base excision repair (BER). Targeting BRCA1-BER deficient cells by blockade of ATM and DNA-PKcs could be a promising strategy in breast cancer. We investigated BRCA1, XRCC1 and pol β protein expression in two cohorts ( n = 1602 sporadic and n = 50 germ-line BRCA1 mutated) and mRNA expression in two cohorts ( n = 1952 and n = 249). Artificial neural network analysis for BRCA1-DNA repair interacting genes was conducted in 249 tumours. Pre-clinically, BRCA1 proficient and deficient cells were DNA repair expression profiled and evaluated for synthetic lethality using ATM and DNA-PKcs inhibitors either alone or in combination with cisplatin. In human tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol β at mRNA and protein levels ( p < 0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol β expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol β expressing BRCA1 negative tumours (ps < 0.05). Pre-clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol β. BRCA1-BER deficient cells were sensitive to ATM and DNA-PKcs inhibitor treatment either alone or in combination with cisplatin and synthetic lethality was evidenced by DNA double strand breaks accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol β expression status in BRCA1 negative tumours may have prognostic significance. BRCA1-BER deficient cells could be targeted by ATM or DNA-PKcs inhibitors for personalized therapy. [ABSTRACT FROM AUTHOR]

Stathmin is a p53-target gene, frequently overexpressed in late stages of human cancer progression. Type II High Grade Epithelial Ovarian Carcinomas (HG-EOC) represents the only clear exception to this observation. Here, we show that stathmin expression is necessary for the survival of HG-EOC cells carrying a p53 mutant (p53MUT) gene. At molecular level, stathmin favours the binding and the phosphorylation of p53MUT by DNA-PKCS, eventually modulating p53MUT stability and transcriptional activity. Inhibition of stathmin or DNA-PKCS impaired p53MUT-dependent transcription of several M phase regulators, resulting in M phase failure and EOC cell death, both in vitro and in vivo. In primary human EOC a strong correlation exists between stathmin, DNA-PKCS, p53MUT overexpression and its transcriptional targets, further strengthening the relevance of the new pathway here described. Overall our data support the hypothesis that the expression of stathmin and p53 could be useful for the identification of high risk patients that will benefit from a therapy specifically acting on mitotic cancer cells. [ABSTRACT FROM AUTHOR]

Poly-ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia-telangiectasia mutated (ATM)-deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM-deficient MCL cells, olaparib induced DNA-PK-dependent phosphorylation and stabilization of p53 as well as expression of p53-responsive cell cycle checkpoint regulators, and inhibition of DNA-PK reduced the toxicity of olaparib in ATM-deficient MCL cells. Thus, both DNA-PK and p53 regulate the response of ATM-deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53-deficient malignancies. [ABSTRACT FROM AUTHOR]

Defects in the DNA damage response pathway [e.g. del(17p)] are associated with drug-resistant B-cell chronic lymphocytic leukaemia (CLL). We previously demonstrated that over-expression of DNA-dependent protein kinase (DNA-PK) correlates with chemo-resistance and that inhibition of DNA-PK sensitizes CLL cells to chemotherapeutics. Here, we investigated expression of DNA-PK and other proteins that impact on drug resistance, and evaluated the effects of a DNA-PK inhibitor (NU7441) on mitoxantrone-induced cytotoxicity in CLL cells. NU7441 sensitized cells from 42/49 CLL samples to mitoxantrone, with sensitization ranging from 2- to 200-fold Co-culture of CLL cells in conditioned stromal medium increased chemoresistance but did not reduce sensitization by NU7441. Mitoxantrone treatment induced γH2AX foci and NU7441 increased their longevity (24 h). NU7441 prevented mitoxantrone-induced autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser 2056, confirming that DNA-PK participates in repair of mitoxantrone-induced DNA damage. del(17p) cases were more resistant to mitoxantrone than del(13q) cases, but were resensitized (7-16 fold) by co-incubation with NU7441. Expression of DNA-PKcs, Ku80, P-glycoprotein and topoisomerase IIβ were significantly higher in del(17p) cases. PRKDC mRNA levels correlated with DNA-PKcs protein expression, which predicted shorter survival. These data confirm the potential of DNA-PK as a therapeutic target in poor prognosis CLL. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol-3 kinase-related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C-terminal regions, referred to as PIKK-regulatory domain (PRD) and FRAP-ATM-TRRAP-C-terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol-3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C-terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop. [ABSTRACT FROM AUTHOR]

Hypoxic preconditioning may afford protection against subsequent lethal hypoxia. As hypoxic tolerance induces changes in the expression of genes involved in DNA damage and repair response pathways, we investigated whether DNA-dependent protein kinase (DNA-PK), one of the DNA double-strand break repair proteins, could be involved in hypoxic preconditioning-induced protective signaling cascades. We showed that induction of hypoxia-inducible factor-1α expression during hypoxic preconditioning by repeated hypoxic exposure was associated with increased mRNA and protein levels of DNA-PK catalytic subunit (DNA-PKcs) and Ku70/Ku80, the DNA-PK components, in human hepatoma HepG2 cells, followed by upregulation of Hsp70/Hsp90 and Bcl-2 and concurrent downregulation of Bax. Additionally, loss of DNA-PKcs led to attenuated expression of Hsp70/Hsp90, accelerated hypoxia-inducible factor-1α degradation, and increased susceptibility to hypoxia-induced cell death. We also found that the mRNA and protein levels of heat shock factor-1 (HSF1) were progressively increased with DNA-PK activation during hypoxic preconditioning, and inhibition of HSF1 function by KNK437 resulted in a significant decrease in the level of protein kinase Akt as well as of DNA-PKcs, with downregulation of Hsp70/Hsp90 and HIF-1α. Our results suggest the possibility that DNA-PK-mediated signaling pathway is required for the increase in HIF-1α expression through activation of HSF1 and subsequent upregulation of heat shock proteins after hypoxic reconditioning. [ABSTRACT FROM AUTHOR]

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609–T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing. [ABSTRACT FROM AUTHOR]

The occurrence of DNA double-strand breaks in the nucleus provokes in its structural organization a large-scale alteration whose molecular basis is still mostly unclear. Here, we show that double-strand breaks trigger preferential assembly of nucleoproteins in human cellular fractions and that they mediate the separation of large protein–DNA aggregates from aqueous solution. The interaction among the aggregative nucleoproteins presents a dynamic condition that allows the effective interaction of nucleoproteins with external molecules like free ATP and facilitates intrinsic DNA end-joining activity. This aggregative organization is functionally coacervate-like. The key component is DNA-dependent protein kinase (DNA-PK), which can be characterized as a DNA-specific aggregation factor as well as a nuclear scaffold/matrix-interactive factor. In the context of aggregation, the kinase activity of DNA-PK is essential for efficient DNA end-joining. The massive and functional concentration of nucleoproteins on DNA in vitro may represent a possible status of nuclear dynamics in vivo, which probably includes the DNA-PK-dependent response to multiple double-strand breaks. [ABSTRACT FROM AUTHOR]

Abstract: The viability of non-homologous end-joining (NHEJ)-defective mice suggests that homologous recombination (HR) might take over its role in DNA repair. To test this hypothesis, we examined gene targeting frequencies (TF) in DNA-PKcs , Ku80 and poly(ADP-ribose) polymerase (PARP-1) nullizygous cells. We observed a 3-fold TF increase in PARP-1 knockout embryonic stem (ES) cells, which is consistent with the predicted role of PARP-1 as a switch between HR and NHEJ. To a lesser extent, such effect could be reproduced upon chemical inhibition of PARP-1. However, TF was not enhanced in Ku80- or DNA-PKcs -defective cells. Our study also suggests an unexpected involvement of DNA-PKcs in HR. [Copyright &y& Elsevier]

The DNA-dependent protein kinase (DNA-PK) is a trimeric factor originally identified as an enzyme that becomes activated upon incubation with DNA. Genetic defects in either the catalytic subunit (DNA-PKCS) or the two Ku components of DNA-PK result in immunodeficiency, radiosensitivity, and premature aging. This combined phenotype is generally attributed to the requirement for DNA-PK in the repair of DNA double strand breaks during various biological processes. However, recent studies revealed that DNA-PKCS a member of the growing family of phosphatidylinositol 3-kinases, participates in signal transduction cascades related to apoptotic cell death, telomere maintenance and other pathways of genome surveillance. These manifold functions of DNA-PKCS have been associated with an increasing number of protein interaction partners and phosphorylation targets. Here we review the DNA binding properties of DNA-PKCS and highlight its ability to interact with an astounding diversity of nucleic acid substrates. This survey indicates that the large catalytic subunit of DNA-PK functions as a sensor of not only broken DNA molecules, but of a wider spectrum of aberrant, unusual, or specialized structures that interrupt the standard double helical conformation of DNA. [ABSTRACT FROM AUTHOR]

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death. [ABSTRACT FROM AUTHOR]

Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation. NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal. [ABSTRACT FROM AUTHOR]

We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line. The enzyme removes 5' single-strand extensions from duplex DNA molecules. The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase. The exonuclease activity does not require divalent cations. The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed. [ABSTRACT FROM AUTHOR]

We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C-terminal deletion mutants, which contain so-called Mdm2-binding sites, is markedly decreased, compared with that of wild-type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53 s tertiary structure and/or C-terminal region may also be important for this reaction. DNA-dependent protein kinase is known to phosphorylate p53 on Mdm2-binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage-induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19ARF affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19ARF-bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19ARF promotes the stabilization of p53 by inactivating Mdm2. [ABSTRACT FROM AUTHOR]

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase. [ABSTRACT FROM AUTHOR]

DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair. One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit. We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy. Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku. Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK. [ABSTRACT FROM AUTHOR]