Frameshift Mutation in<i>PRKDC</i>, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J

Anderson, Carl W.; Dunn, John J.; Freimuth, Paul; Galloway, Anne M.; Allalunis-Turner, M. J.

doi:10.1667/0033-7587(2001)156[0002:fmiptg]2.0.co;2

Cited by 63 publications

(34 citation statements)

References 41 publications

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“…We therefore performed immunostaining experiments with the matched human glioblastoma cell lines M059J and M059K (2). M059J lacks DNA-PKcs and is radiation sensitive, whereas M059K is its DNA-PKcs-containing, radiationresistant counterpart (3,36). Control immunostaining experiments confirmed the presence of DNA-PKcs in our cultures of M059K cells and its absence from M059J cells, consistent with published reports (data not shown).…”

Section: Resultssupporting

confidence: 92%

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Dynan

2002

Molecular and Cellular Biology

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Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominantnegative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.The Ku protein is part of the DNA-dependent protein kinase (DNA-PK), which is required for the nonhomologous end-joining pathway of DNA double-strand break (DSB) repair (53, 59). When a break occurs, Ku binds avidly to the DNA ends, translocates inward, and recruits the 470-kDa DNA-PK catalytic subunit (DNA-PKcs) to form an active protein kinase complex (19,26,74). Proteins in this complex cooperate with XRCC4, DNA ligase IV, and other factors to carry out DSB repair (reviewed in reference 22). Ku-deficient cells are sensitive to ionizing radiation and are unable to complete the process of V(D)J recombination, which involves a DSB intermediate.In addition to its well-documented role in DSB repair, Ku appears to have some interaction with the RNA polymerase II (RNAP II) transcription apparatus. Several reports describe the binding of Ku to promoter regions or its ability to regulate transcription of individual genes (reviewed in reference 21 and also see references 11, 25, 31, and 72). Other reports describe interaction of Ku with the general transcription machinery. DNA-PK efficiently phosphorylates RNAP II in vitro (50), and a fraction of Ku resides in RNAP II-containing complexes (41). The importance of these interactions is suggested by the finding that nuclear extracts of Ku-deficient cells exhibit a characteristic transcription defect (70). Transcription of several different promoters is decreased two-to fivefold relative to that of extracts from a matched Ku-containing cell line (70). The defect is entirely at the level of reinitiation and is not seen in assays in which transcription is limited to a ...

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Section: Resultssupporting

confidence: 92%

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Dynan

2002

Molecular and Cellular Biology

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“…1D). Phosphorylation of p53 on Ser 20 and Ser 37 was also similarly positive in all lines (data not shown). In addition to immunoblotting, immunostaining revealed augmentation of H2AX phosphorylation on Ser 139 in all lines, as manifested by nuclear foci, which was more remarkable in ATM-repleted cells (Supplementary Fig.…”

Section: Activation Of Cndac-induced G 2 Checkpoint Is Independent Ofmentioning

confidence: 62%

Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosylcytosine

Liu

Matsuda

Plunkett

2008

Molecular Cancer Therapeutics

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Abstract2 ¶-C-Cyano-2 ¶-deoxy-1-B-D-arabino-pentofuranosylcytosine (CNDAC), the prodrug (sapacitabine) of which is in clinical trials, has the novel mechanism of action of causing single-strand breaks after incorporating into DNA. Cells respond to this unique lesion by activating the G 2 checkpoint, affected by the Chk1-Cdc25C-cyclindependent kinase 1/cyclin B pathway. This study aims at defining DNA damage checkpoint sensors that activate this response to CNDAC, particularly focusing on the major phosphatidylinositol 3-kinase -like protein kinase family proteins. First, fibroblasts, deficient in ataxiatelangiectasia mutated (ATM), transfected with empty vector or repleted with ATM, were arrested in G 2 by CNDAC to similar extents, suggesting ATM is not required to activate the G 2 checkpoint. Second, chromatin associations of RPA70 and RPA32, subunits of the ssDNAbinding protein, and the ataxia-telangiectasia and Rad3-related (ATR) substrate Rad17 and its phosphorylated form were increased on CNDAC exposure, suggesting activation of ATR kinase. The G 2 checkpoint was abrogated due to depletion of ATR by small interfering RNA, and impaired in ATR-Seckel cells, indicating participation of ATR in this G 2 checkpoint pathway. Third, the G 2 checkpoint was more stringent in glioma cells with wild-type DNA-dependent protein kinase catalytic subunit (DNA-PKcs) than those with mutant DNA-PKcs, as shown by mitotic index counting. CNDAC-induced G 2 arrest was abrogated by specific DNA-PKcs inhibitors or small interfering RNA knockdown in ML-1 and/or HeLa cells. Finally, two phosphatidylinositol 3-kinase -like protein kinase inhibitors, caffeine and wortmannin, abolished the CNDAC-induced G 2 checkpoint in a spectrum of cell lines. Together, our data showed that ATR and DNA-PK cooperate in CNDAC-induced activation of the G 2 checkpoint pathway.

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“…1A). The elevated number of foci in these glioma cell lines relative to primary cell lines is most likely explained by the fact that the cells are hyperdiploid (modal chromosome number approximately 110), allowing the formation of more DSBs (34). Foci both form and commence disappearing more rapidly in the M059K (DNA-PKcs ϩ/ϩ ) cells relative to the fibroblasts.…”

Section: Methodsmentioning

confidence: 94%

ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation

Stiff

O’Driscoll²,

Rief³

et al. 2004

Cancer Research

918

668

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H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.

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Frameshift Mutation inPRKDC, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J

Cited by 63 publications

References 41 publications

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosylcytosine

ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation

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