ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress

Yajima, H.; Lee, Kyung Jong; Chen, Benjamin P.C.

doi:10.1128/mcb.00048-06

Cited by 110 publications

(133 citation statements)

References 37 publications

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“…In addition, Nu7441 but not the ATM kinase inhibitor Ku55933 attenuated mitotic DNA-PKcs phosphorylation at Thr-2609. We have reported previously that DNA-PKcs Thr-2609 phosphorylation is mediated by ATM and ATR kinases in response to IR and UV-induced replication stress, respectively (8,9). The result in Fig.…”

Section: Discussionmentioning

confidence: 59%

“…We have previously reported that DNA-PKcs kinase autophosphorylates itself at Ser-2056 in response to IR (7), whereas ATM and ATR kinases are required for DNA-PKcs phosphorylation at Thr-2609 (8,9). To determine whether ATM is involved in mitotic DNA-PKcs phosphorylation, ATM-proficient 1BR3 and deficient AT5 human fibroblasts were arrested in mitosis and were analyzed for DNA-PKcs phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

“…HeLa cell synchronization was carried out by double thymidine blockage at the G 1 /S boundary and released in culture medium containing 50 ng/ml nocodazole for subsequent cell cycle arrest at mitosis (14). Small interfering RNA (siRNA) oligonucleotides designed against DNA-PKcs (15), ATM, and ATR (9) were transfected with RNAiMax (Invitrogen) as described (9).…”

Section: Methodsmentioning

confidence: 99%

“…Whereas IR-induced DNA-PKcs phosphorylation at Ser-2056 is clearly mediated by the autophosphorylation of DNA-PKcs (7), IR-induced DNAPKcs phosphorylation at the Thr-2609 cluster is mainly dependent on ataxia telangiectasia mutated (ATM) kinase but not DNA-PKcs itself (8). In addition, the Thr-2609 cluster region can be phosphorylated by ATR kinase in response to UV irradiation or replication stresses (9). Although the precise mechanism of DNA-PKcs phosphorylation remains to be clarified, like its kinase activity, DNA-PKcs phosphorylation at Ser-2056 and the Thr-2609 cluster are required for DSB repair.…”

mentioning

confidence: 99%

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Involvement of DNA-dependent Protein Kinase in Normal Cell Cycle Progression through Mitosis

Lee

Lin

Chou

et al. 2011

Journal of Biological Chemistry

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The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the nonhomologous end joining pathway. When DSBs occur, DNAPKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNAPKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNAPKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation. DNA-PKcs,3 the catalytic subunit of DNA-dependent protein kinase (DNA-PK), is known to play an essential role in non-homologous end joining-mediated DNA double-strand break (DSB) repair in mammalian cells. In response to DSBs, the Ku70/80 subunits of DNA-PK immediately bind to the broken DSB ends and subsequently recruit and activate DNA-PKcs kinase activity (1). The intrinsic kinase activity of DNA-PKcs is essential for its role in DSB repair (2), likely through phosphorylation and regulation of non-homologous end joining components, including DNA-PKcs itself. DNA-PKcs is rapidly autophosphorylated in vitro upon activation and is also phosphorylated in vivo after exposure to ionizing radiation (IR). Among all the phosphorylation sites identified (3-6), phosphorylation in vivo was clearly detected at the Ser-2056 residue and at the Thr-2609 cluster region (3,7,8). Whereas IR-induced DNA-PKcs phosphorylation at Ser-2056 is clearly mediated by the autophosphorylation of DNA-PKcs (7), IR-induced DNAPKcs phosphorylation at the Thr-2609 cluster is mainly dependent on ataxia telangiectasia mutated (ATM) kinase but not DNA-PKcs itself (8). In addition, the Thr-2609 cluster region can be phosphorylated by ATR kinase in response to UV irradiation or replication stresses (9). Although the precise mechanism of DNA-PKcs phosphorylation remains to be clarified, like its kinase activity, DNA-PKcs phosphorylation at Ser-2056 and the Thr-2609 cluster are required for DSB repair.In addition to its roles in DNA damage response, DNA-PKcsmediated DSB repair is required for V(D)J recombination during T-and B-cell maturation (10). Recently, it has also been reported that DNA-PKcs phosphorylates and activates the upstream stimulatory factor, which in turn regulates the expression of fatty acid synthase in response to feeding (11). In contrast to the well studied role of DNA-PKcs in DSB repair, not much is known about the involvement of DNA-PKcs kinase and its phosphorylations in other cellular activities, particularly under normal conditions. While an...

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Section: Discussionmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Involvement of DNA-dependent Protein Kinase in Normal Cell Cycle Progression through Mitosis

Lee

Lin

Chou

et al. 2011

Journal of Biological Chemistry

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“…Based on this, it is likely that DNA-PK, which normally regulates repair of DSBs by NHEJ, also responds to DSBs induced during replication stress. It has been previously reported that DNA-PK undergoes auto-phosphorylation after the treatment of cells with genotoxic agents such as UV, hydroxyurea and the TOP1 inhibitor, camptothecin 15,16 . In addition, DNA-PK becomes rapidly activated following nucleotide depletion when Chk1 activity is compromised 17 .…”

Section: Alchemix Induces a Marked Atr-dependent Dna Damage Response mentioning

confidence: 99%

The dual-acting chemotherapeutic agent Alchemix induces cell death independently of ATM and p53

et al. 2014

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Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response genes, such as ATM and TP53. Here, we characterize the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DNA damage response at nano-molar concentrations and this is mediated primarily through ATR-and DNA-PK-but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model.Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DNA damage response genes.

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Non‐homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process

Rulten

Grundy

2017

BioEssays

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Non-homologous end-joining (NHEJ) is the dominant means of repairing chromosomal DNA double strand breaks (DSBs), and is essential in human cells. Fifteen or more proteins can be involved in the detection, signalling, synapsis, end-processing and ligation events required to repair a DSB, and must be assembled in the confined space around the DNA ends. We review here a number of interaction points between the core NHEJ components (Ku70, Ku80, DNA-PKcs, XRCC4 and Ligase IV) and accessory factors such as kinases, phosphatases, polymerases and structural proteins. Conserved protein-protein interaction sites such as Ku-binding motifs (KBMs), XLF-like motifs (XLMs), FHA and BRCT domains illustrate that different proteins compete for the same binding sites on the core machinery, and must be spatially and temporally regulated. We discuss how post-translational modifications such as phosphorylation, ADP-ribosylation and ubiquitinylation may regulate sequential steps in the NHEJ pathway or control repair at different types of DNA breaks.

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ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress

Cited by 110 publications

References 37 publications

Involvement of DNA-dependent Protein Kinase in Normal Cell Cycle Progression through Mitosis

Involvement of DNA-dependent Protein Kinase in Normal Cell Cycle Progression through Mitosis

The dual-acting chemotherapeutic agent Alchemix induces cell death independently of ATM and p53

Non‐homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process

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