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David J. Chen - One of the best experts on this subject based on the ideXlab platform.
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Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance
Biochemical and biophysical research communications, 2016Co-Authors: Eiichiro Mori, Anthony J. Davis, Hasegawa Masatoshi, David J. ChenAbstract:DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs.
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BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle
Nucleic acids research, 2014Co-Authors: Anthony J. Davis, Eiichiro Mori, Linfeng Chi, Kyung Jong Lee, Kazi R. Fattah, Jun Yang, David J. ChenAbstract:Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.
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ku and dna dependent protein kinase dynamic conformations and assembly regulate dna binding and the initial non homologous end joining complex
Journal of Biological Chemistry, 2010Co-Authors: Michal Hammel, David J. Chen, Brandi L Mahaney, Brandon Cai, Barry M Phipps, Robert P Rambo, Greg L Hura, Martin Pelikan, Ramin M Abolfath, Susan P LeesmillerAbstract:DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.
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DNA-PKcs and ATM co-regulate DNA double-strand break repair
DNA repair, 2009Co-Authors: Meena Shrivastav, Benjamin P C Chen, David J. Chen, Cheryl A. Miller, Leyma P. De Haro, Stephen T. Durant, Jac A. NickoloffAbstract:DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ∼4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.
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DNA-PKcs-Dependent Modulation of Cellular Radiosensitivity by a Selective Cyclooxygenase-2 Inhibitor
International journal of radiation oncology biology physics, 2007Co-Authors: Elisabeth Kodym, Benjamin P C Chen, David J. Chen, Keiko Morotomi-yano, Reinhard Kodym, Hak Choy, Debabrata SahaAbstract:Purpose Inhibition of cyclooxygenase-2 has been shown to increase radiosensitivity. Recently, the suppression of radiation-induced DNA-dependant protein kinase (DNA-PK) activity by the selective cyclooxygenase-2 inhibitor celecoxib was reported. Given the importance of DNA-PK for repair of radiation-induced DNA double-strand breaks by nonhomologous end-joining and the clinical use of the substance, we investigated the relevance of the DNA-PK catalytic subunit (DNA-PKcs) for the modulation of cellular radiosensitivity by celecoxib. Methods and Materials We used a syngeneic model of Chinese hamster ovarian cell lines: AA8, possessing a wild-type DNK-PKcs; V3, lacking a functional DNA-PKcs; and V3/WT11, V3 stably transfected with the DNA-PKcs. The cells were treated with celecoxib (50 μM) for 24 h before irradiation. The modulation of radiosensitivity was determined using the colony formation assay. Results Treatment with celecoxib increased the cellular radiosensitivity in the DNA-PKcs-deficient cell line V3 with a dose-enhancement ratio of 1.3 for a surviving fraction of 0.5. In contrast, clonogenic survival was increased in DNA-PKcs wild-type–expressing AA8 cells and in V3 cells transfected with DNA-PKcs (V3/WT11). The decrease in radiosensitivity was comparable to the radiosensitization in V3 cells, with a dose-enhancement ratio of 0.76 (AA8) and 0.80 (V3/WT11) for a survival of 0.5. Conclusions We have demonstrated a DNA-PKcs–dependent differential modulation of cellular radiosensitivity by celecoxib. These effects might be attributed to alterations in signaling cascades downstream of DNA-PK toward cell survival. These findings offer an explanation for the poor outcomes in some recently published clinical trials.
Benjamin P C Chen - One of the best experts on this subject based on the ideXlab platform.
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PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway.
Nucleic acids research, 2018Co-Authors: Yu Fen Lin, Zengfu Shang, Hung Ying Shih, Ching-te Kuo, Jiaming Guo, Hsinyu Lee, Benjamin P C ChenAbstract:The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N' terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.
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DNA-PKcs activates the Chk2-Brca1 pathway during mitosis to ensure chromosomal stability (736.4)
The FASEB Journal, 2014Co-Authors: Benjamin P C Chen, Zengfu ShangAbstract:and BPC ChenThe catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is known to have a critical role in DNA double-strand breakrepair. We have previously reported that DNA-PKcs is activated when cells enter mitosis and functions in mitotic spindle assemblyand chromosome segregation. Here we report that DNA-PKcs is the upstream regulator of the Chk2–Brca1 pathway, which impactsmicrotubule dynamics, kinetochore attachment and chromosomal segregation in mitosis. Downstream from Chk2, Brca1 promotesmonoubiquitination of g-tubulin to inhibit microtubule nucleation and growth. We found that DNA-PKcs is essential for mitoticChk2 phosphorylation at Thr68. As in Chk2- and Brca1-deficient cells, loss of DNA-PKcs resulted in chromosome misalignment andlagging during anaphase owing to elevation in microtubule dynamics. Importantly, these mitotic aberrations in DNA-PKcs-defectivecells were alleviated by the overexpression of phosphomimetic Chk2 or Brca1 mutant proteins but not their wild-type counterparts.Taken together, these results demonstrate that DNA-PKcs regulates mitotic spindle organization and chromosomal instability viathe Chk2–Brca1 signaling pathway.Oncogenesis (2014) 3, e85; doi:10.1038/oncsis.2013.49; published online 3 February 2014Subject Categories: Cell cycle and growth regulationKeywords: DNA-PKcs; Chk2; Brca1; chromosomal instability; microtubuleINTRODUCTIONChromosome instability (CIN), characterized by increased fre-quency of gain or loss of the whole chromosomes, has long beenimplicated in tumorigenesis, poor patient prognosis and drugresistance.
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DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response
Nucleic acids research, 2014Co-Authors: Yu Fen Lin, Zengfu Shang, Hung Ying Shih, Shinji Matsunaga, Benjamin P C ChenAbstract:The ataxia telangiectasia mutated and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis is the major signaling pathway activated in response to replication stress and is essential for the intra-S checkpoint. ATR phosphorylates and activates a number of molecules to coordinate cell cycle progression. Chk1 is the major effector downstream from ATR and plays a critical role in intra-S checkpoint on replication stress. Activation of Chk1 kinase also requires its association with Claspin, an adaptor protein essential for Chk1 protein stability, recruitment and ATR-dependent Chk1 phosphorylation. We have previously reported that, on replication stress, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated by ATR at the stalled replication forks and is required for cellular resistance to replication stresses although the impact of DNA-PKcs onto the ATR signaling pathway remains elusive. Here we report that ATR-dependent Chk1 phosphorylation and Chk1 signaling are compromised in the absence of DNA-PKcs. Our investigation reveals that DNA-PKcs is required to maintain Chk1–Claspin complex stability and transcriptional regulation of Claspin expression. The impaired Chk1 activity results in a defective intra-S checkpoint response in DNA-PKcs–deficient cells. Taken together, these results suggest that DNA-PKcs, in addition to its direct role in DNA damage repair, facilitates ATR-Chk1 signaling pathway in response to replication stress.
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DNA-PKcs activates the Chk2-Brca1 pathway during mitosis to ensure chromosomal stability.
Oncogenesis, 2014Co-Authors: Z Shang, C.y Shen, S. Matsunaga, Y. F. Lin, Benjamin P C ChenAbstract:The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is known to have a critical role in DNA double-strand break repair. We have previously reported that DNA-PKcs is activated when cells enter mitosis and functions in mitotic spindle assembly and chromosome segregation. Here we report that DNA-PKcs is the upstream regulator of the Chk2–Brca1 pathway, which impacts microtubule dynamics, kinetochore attachment and chromosomal segregation in mitosis. Downstream from Chk2, Brca1 promotes monoubiquitination of γ-tubulin to inhibit microtubule nucleation and growth. We found that DNA-PKcs is essential for mitotic Chk2 phosphorylation at Thr68. As in Chk2- and Brca1-deficient cells, loss of DNA-PKcs resulted in chromosome misalignment and lagging during anaphase owing to elevation in microtubule dynamics. Importantly, these mitotic aberrations in DNA-PKcs-defective cells were alleviated by the overexpression of phosphomimetic Chk2 or Brca1 mutant proteins but not their wild-type counterparts. Taken together, these results demonstrate that DNA-PKcs regulates mitotic spindle organization and chromosomal instability via the Chk2–Brca1 signaling pathway.
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DNA-PKcs and ATM co-regulate DNA double-strand break repair
DNA repair, 2009Co-Authors: Meena Shrivastav, Benjamin P C Chen, David J. Chen, Cheryl A. Miller, Leyma P. De Haro, Stephen T. Durant, Jac A. NickoloffAbstract:DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ∼4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.
Tanya T Paull - One of the best experts on this subject based on the ideXlab platform.
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Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM
Molecular cell, 2016Co-Authors: Yi Zhou, Wenxia Jiang, Jennifer L Crowe, Ji-hoon Lee, Shan Zha, Tanya T PaullAbstract:Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.
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dna dependent protein kinase regulates dna end resection in concert with mre11 rad50 nbs1 mrn and ataxia telangiectasia mutated atm
Journal of Biological Chemistry, 2013Co-Authors: Yi Zhou, Tanya T PaullAbstract:Abstract The resection of DNA double strand breaks (DSBs) initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic non-homologous end joining (NHEJ) factor, antagonizes DSB resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. ATM kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11/Rad50/Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and also inhibits DNA Ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in NHEJ, DNA-PKcs also acts in concert with MRN and ATM to regulate resection and thus DNA repair pathway choice.
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dna dependent protein kinase regulates dna end resection in concert with mre11 rad50 nbs1 mrn and ataxia telangiectasia mutated atm
Journal of Biological Chemistry, 2013Co-Authors: Yi Zhou, Tanya T PaullAbstract:The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.
Zengfu Shang - One of the best experts on this subject based on the ideXlab platform.
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PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway.
Nucleic acids research, 2018Co-Authors: Yu Fen Lin, Zengfu Shang, Hung Ying Shih, Ching-te Kuo, Jiaming Guo, Hsinyu Lee, Benjamin P C ChenAbstract:The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N' terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.
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DNA‐PKcs Associates With PLK1 and Is Involved in Proper Chromosome Segregation and Cytokinesis
Journal of cellular biochemistry, 2014Co-Authors: Bo Huang, Zengfu Shang, Shimeng Zhang, Yu Wang, Hua Guan, Xiao-dan Liu, Weiqing Rang, Ping-kun ZhouAbstract:Accurate mitotic regulation is as important as intrinsic DNA repair for maintaining genomic stability. It is believed that these two cellular mechanisms are interconnected with DNA damage. DNA-PKcs is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair, and it was recently discovered to be involved in mitotic processing. However, the underlying mechanism of DNA-PKcs action in mitotic control is unknown. Here, we demonstrated that depletion of DNA-PKcs led to the dysregulation of mitotic progression in response to DNA damage, which eventually resulted in multiple failures, including failure to segregate sister chromatids and failure to complete cytokinesis, with daughter cells becoming fused again. The depletion of DNA-PKcs resulted in a notable failure of cytokinesis, with a high incidence of multinucleated cells. There were also cytoplasmic bridges containing DNA that continuously connected the daughter cells after DNA damage was induced. Phosphorylated DNA-PKcs (T2609) colocalizes with PLK1 throughout mitosis, including at the centrosomes from prophase to anaphase and at the kinetochores from prometaphase to metaphase, with accumulation at the midbody during cytokinesis. Importantly, DNA-PKcs was found to associate with PLK1 in the mitotic phase, and the depletion of DNA-PKcs resulted in the overexpression of PLK1 due to increased protein stability. However, deficiency in DNA-PKcs attenuated the recruitment of phosphorylated PLK1 to the midbody but not to the kinetochores and centrosomes. Our results demonstrate the functional association of DNA-PKcs with PLK1, especially in chromosomal segregation and cytokinesis control. J. Cell. Biochem. 115: 1077–1088, 2014. © 2013 Wiley Periodicals, Inc.
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DNA-PKcs activates the Chk2-Brca1 pathway during mitosis to ensure chromosomal stability (736.4)
The FASEB Journal, 2014Co-Authors: Benjamin P C Chen, Zengfu ShangAbstract:and BPC ChenThe catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is known to have a critical role in DNA double-strand breakrepair. We have previously reported that DNA-PKcs is activated when cells enter mitosis and functions in mitotic spindle assemblyand chromosome segregation. Here we report that DNA-PKcs is the upstream regulator of the Chk2–Brca1 pathway, which impactsmicrotubule dynamics, kinetochore attachment and chromosomal segregation in mitosis. Downstream from Chk2, Brca1 promotesmonoubiquitination of g-tubulin to inhibit microtubule nucleation and growth. We found that DNA-PKcs is essential for mitoticChk2 phosphorylation at Thr68. As in Chk2- and Brca1-deficient cells, loss of DNA-PKcs resulted in chromosome misalignment andlagging during anaphase owing to elevation in microtubule dynamics. Importantly, these mitotic aberrations in DNA-PKcs-defectivecells were alleviated by the overexpression of phosphomimetic Chk2 or Brca1 mutant proteins but not their wild-type counterparts.Taken together, these results demonstrate that DNA-PKcs regulates mitotic spindle organization and chromosomal instability viathe Chk2–Brca1 signaling pathway.Oncogenesis (2014) 3, e85; doi:10.1038/oncsis.2013.49; published online 3 February 2014Subject Categories: Cell cycle and growth regulationKeywords: DNA-PKcs; Chk2; Brca1; chromosomal instability; microtubuleINTRODUCTIONChromosome instability (CIN), characterized by increased fre-quency of gain or loss of the whole chromosomes, has long beenimplicated in tumorigenesis, poor patient prognosis and drugresistance.
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DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response
Nucleic acids research, 2014Co-Authors: Yu Fen Lin, Zengfu Shang, Hung Ying Shih, Shinji Matsunaga, Benjamin P C ChenAbstract:The ataxia telangiectasia mutated and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis is the major signaling pathway activated in response to replication stress and is essential for the intra-S checkpoint. ATR phosphorylates and activates a number of molecules to coordinate cell cycle progression. Chk1 is the major effector downstream from ATR and plays a critical role in intra-S checkpoint on replication stress. Activation of Chk1 kinase also requires its association with Claspin, an adaptor protein essential for Chk1 protein stability, recruitment and ATR-dependent Chk1 phosphorylation. We have previously reported that, on replication stress, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated by ATR at the stalled replication forks and is required for cellular resistance to replication stresses although the impact of DNA-PKcs onto the ATR signaling pathway remains elusive. Here we report that ATR-dependent Chk1 phosphorylation and Chk1 signaling are compromised in the absence of DNA-PKcs. Our investigation reveals that DNA-PKcs is required to maintain Chk1–Claspin complex stability and transcriptional regulation of Claspin expression. The impaired Chk1 activity results in a defective intra-S checkpoint response in DNA-PKcs–deficient cells. Taken together, these results suggest that DNA-PKcs, in addition to its direct role in DNA damage repair, facilitates ATR-Chk1 signaling pathway in response to replication stress.
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PIG3 Functions in DNA Damage Response through Regulating DNA-PKcs Homeostasis
International journal of biological sciences, 2013Co-Authors: Zengfu Shang, Shimeng Zhang, Yu Wang, Hua Guan, Xiao-dan Liu, Jiao Jiao Yin, Ping-kun ZhouAbstract:The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnormal DNA damage response, including decreased IR-induced phosphorylation of H2AX, Chk1, Chk2 and Kap-1 as well as a prolonged G2-M arrest and aberrant mitotic progression. Notably, PIG3 knockdown resulted in a striking depression of cellular DNA-PKcs protein level, and was accompanied by a downregulation of ATM. Re-expression of PIG3 effectively rescued the depression of DNA-PKcs in PIG3-depleted cells. This negative regulation of DNA-PKcs by depleting PIG3 seemed to take place at the translational level but not at the levels of transcription or protein degradation. However, a compensatory feedback of increased mRNA expression of DNA-PKcs was formed in PIG3-depleted cells after a few passages or cell cycles of subculture, which led the recovery of the DNA-PKcs protein level and the consequent recovered efficiency of the DNA damage response. These results provide a new insight into the mechanism of PIG3's functioning in DNA damage signaling and the regulation network of cellular DNA-PKcs expression homeostasis.
Susan P. Lees-miller - One of the best experts on this subject based on the ideXlab platform.
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Established and Emerging Roles of the DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs)
Cancer Drug Discovery and Development, 2018Co-Authors: Edward Bartlett, Susan P. Lees-millerAbstract:The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a large polypeptide of over 4000 amino acids with serine/threonine protein kinase activity that is enhanced in the presence of double stranded DNA and the Ku70/80 heterodimer. The discovery of this DNA activated protein kinase activity led to investigation of its role in DNA double-strand break repair and DNA-PKcs was shown to play important roles in repair of ionizing radiation-induced DNA double strand breaks and V(D)J recombination through the non-homologous end joining (NHEJ) pathway. However, recently, additional roles for DNA-PKcs in mitosis, transcription and cell migration have been suggested. Here, we review the structure, established and emerging roles of DNA-PKcs and its potential as a target for cancer therapy.
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A structural model for regulation of NHEJ by DNA-PKcs autophosphorylation
DNA repair, 2010Co-Authors: Tracey A. Dobbs, John A. Tainer, Susan P. Lees-millerAbstract:The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku heterodimer together form the biologically critical DNA-PK complex that plays key roles in the repair of ionizing radiation-induced DNA double-strand breaks through the non-homologous end-joining (NHEJ) pathway. Despite elegant and informative electron microscopy studies, the mechanism by which DNA-PK co-ordinates the initiation of NHEJ has been enigmatic due to limited structural information. Here, we discuss how the recently described small angle X-ray scattering structures of full-length Ku heterodimer and DNA-PKcs in solution, combined with a breakthrough DNA-PKcs crystal structure, provide significant insights into the early stages of NHEJ. Dynamic structural changes associated with a functionally important cluster of autophosphorylation sites play a significant role in regulating the dissociation of DNA-PKcs from Ku and DNA. These new structural insights have implications for understanding the formation and control of the DNA-PK synaptic complex, DNA-PKcs activation and initiation of NHEJ. More generally, they provide prototypic information for the phosphatidylinositol-3 kinase-like (PIKK) family of serine/threonine protein kinases that includes Ataxia Telangiectasia-Mutated (ATM) and ATM-, Rad3-related (ATR) as well as DNA-PKcs.
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Protein-DNA complexes containing DNA-dependent protein kinase in crude extracts from human and rodent cells.
Radiation Research, 1999Co-Authors: Nicholas S. Y. Ting, Lauri G. Lintott, Doug W. Chan, Joan Allalunis-turner, Susan P. Lees-millerAbstract:: The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit (DNA-PKcs) and a DNA-binding protein, Ku. Cells lacking DNA-PK activity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination. Although much information regarding the interactions of Ku with DNA ends is available, relatively little is known about the interaction of DNA-PKcs with DNA-bound Ku. Here we show, using electrophoretic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in extracts from cells of normal human cell lines. Extracts from cells of the radiosensitive human cell line M059J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs protein restores complex formation. This assay may be useful for screening for DNA-PK function in cells of human cell lines and for identifying proteins that interact with the DNA-PK-DNA complex. We also show that Ku protein in rodent cells can interact with human DNA-PKcs; however, this assay may be less useful for studying Ku/DNA-PKcs interactions in cells of rodent cell lines due to the low abundance of DNA-PKcs in these cells.