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A role for DNA-PKcs in G2 checkpoint response and DNA end resection after exposure to ionizing radiation

Dueva, Rositsa

DNA damage response (DDR) is a cellular network that comprises signaling from DNA lesions, including DNA double-strand breaks (DSBs), and their repair. ATM and ATR belong to the Phosphatidylinositol-3 kinase-related kinases (PIKK) family and are known as the master controllers of DDR signaling. It is generally accepted that ATM is activated by DSBs or chromatin modifications, while ATR operates in DNA replication and in response to DSBs becomes activated by single-stranded DNA regions at the DSB ends generated by DNA end resection. DNA-PK catalytic subunit (DNA-PKcs), a third member of the PIKK family, is an essential component of the classical non-homologous end joining (c-NHEJ) pathway of DSB repair. However, the catalytic function of DNA-PKcs in c NHEJ has been extensively studied, while its role in DNA damage signaling still remains obscure. In this thesis, we provide evidence of a contribution of DNA-PKcs in the checkpoint response as well, particularly in cells irradiated in S and G2 phases of the cell cycle. The role of DNA-PKcs in this process appears to be specific as this effect is not observed with depletion of KU70/80, an essential factor for recruitment of DNA-PKcs at DSBs. Analysis of G2 arrest and evaluation of the mitotic index using H3pS10 assay after exposure of cells to IR revealed that deficiency of DNA-PKcs or its chemical inactivation is linked with persistent G2 checkpoint; in cells irradiated in S phase this hyper-activated checkpoint is entirely dependent on ATR, while in cells sustaining DNA damage in G2 phase it relies on both ATM/ATR. The requirement of ATR for the prolonged G2 checkpoint in DNA-PKcs deficient background suggests extensive resection at DSB ends which could be visualized by immunostaining of RPA. Hence, the data demonstrate that DNA-PKcs may also contribute to DNA damage response via regulation of DNA end resection. Evaluation of the effects of ATM and ATR kinases on the G2 checkpoint and DNA end resection by using small molecule inhibitors point to a model where ATM, ATR and DNA-PKcs may work as a kinase module to effectively control the G2 checkpoint and the process of resection after exposure to ionizing radiation.

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Dueva, Rositsa: A role for DNA-PKcs in G2 checkpoint response and DNA end resection after exposure to ionizing radiation. 2016.

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