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BMC Molecular Biology (1)
Nucleic Acids Research (1)
Chen, Benjamin PC (2)
Guerra, Barbara (1)
Lin, Yu-Fen (1)
Matsunaga, Shinji (1)
Olsen, Birgitte B (1)
Shang, Zengfu (1)
Shih, Hung-Ying (1)
Svenstrup, Tina H (1)
Wang, Shih-Ya (1)
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DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response
Nucleic Acids Research
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.
Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage
Olsen, Birgitte B
Svenstrup, Tina H
BMC Molecular Biology
The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks.
In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks.
Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.
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