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

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

Shift in G₁-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions

Depletion of HIF-1α by Inducible Cre/loxP Increases the Sensitivity of Cultured Murine Hepatocytes to Ionizing Radiation in Hypoxia

The transcription factor hypoxia-inducible factor (HIF) is the main oxygen sensor which regulates adaptation to cellular hypoxia. The aim of this study was to establish cultured murine hepatocyte derived cells (mHDC) as an in vitro model and to analyze the role of HIF-1α in apoptosis induction, DNA damage repair and sensitivity to ionizing radiation (IR). We have crossed C57/BL6 mice that bear loxP sites flanking exon 2 of Hif1a with mice which carry tamoxifen-inducible global Cre expression. From the offspring, we have established transduced hepatocyte cultures which are permanently HIF-1α deficient after tamoxifen treatment. We demonstrated that the cells produce albumin, acetylcholine esterase, and the cytokeratins 8 and 18 which functionally characterizes them as hepatocytes. In moderate hypoxia, HIF-1α deficiency increased IR-induced apoptosis and significantly reduced the surviving fraction of mHDC as compared to HIF-1α expressing cells in colony formation assays. Furthermore, HIF-1α knockout cells displayed increased IR-induced DNA damage as demonstrated by increased generation and persistence of γH2AX foci. HIF-1α deficient cells showed delayed DNA repair after IR in hypoxia in neutral comet assays which may indicate that non-homologous end joining (NHEJ) repair capacity was affected. Overall, our data suggest that HIF-1α inactivation increases radiation sensitivity of mHDC cells

Increased Resection at DSBs in G₂-Phase Is a Unique Phenotype Associated with DNA-PKcs Defects That Is Not Shared by Other Factors of c-NHEJ

The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 Gy. Increased resection and HR engagement with decreasing DSB-load generate a conundrum in a classical non-homologous end-joining (c-NHEJ)-dominated cell and suggest a mechanism adaptively facilitating resection. We report that ablation of DNA-PKcs causes hyper-resection, implicating DNA-PK in the underpinning mechanism. However, hyper-resection in DNA-PKcs-deficient cells can also be an indirect consequence of their c-NHEJ defect. Here, we report that all tested DNA-PKcs mutants show hyper-resection, while mutants with defects in all other factors of c-NHEJ fail to do so. This result rules out the model of c-NHEJ versus HR competition and the passive shift from c-NHEJ to HR as the causes of the increased resection and suggests the integration of DNA-PKcs into resection regulation. We develop a model, compatible with the results of others, which integrates DNA-PKcs into resection regulation and HR for a subset of DSBs. For these DSBs, we propose that the kinase remains at the break site, rather than the commonly assumed autophosphorylation-mediated removal from DNA ends