DNA-PKcs plays a crucial role in replication stress and oxidative stress responses, and we intend to clarify the mechanisms of how DNA-PKcs assists cellular response to these physiological stresses.
DNA-PKcs participates in cellular response to replication stress when DNA replication forks are attenuated or stalled due to DNA lesions, exhausted deoxyribonucleotides pools (dNTPs), oncogene-driven excessive cell proliferation, etc. Upon UV- or hydroxyurea-generated replication stress, we observed that DNA-PKcs is rapidly recruited to stalled replication forks and phosphorylated by the ATR kinase, which elicits S-phase checkpoint through Chk1 kinase and facilitates recovery of stalled replication forks. In DNA-PKcs defective cells, we observed that there is an increased sensitivity toward UV or hydroxyurea treatments and an impairment in the S-phase checkpoint.
Our investigation revealed that DNA-PKcs could facilitate the ATR signaling pathway through distinctive mechanisms. DNA-PKcs is required to stabilize the Chk1/Claspin complex through transcriptional regulation of Claspin gene expression. In addition, DNA-PKcs interacts directly with the ATR kinase upon replication and facilitates ATR kinase activation. Prolonged replication stalling will lead to collapse of replication forks and DSB formation. How DNA-PKcs participates in the recovery of stalled replication forks and the repair of replication-associated DSBs will be further investigated.
ATM has been shown to be involved in the oxidative stress response and could be activated directly in vitro upon hydrogen peroxide (H2O2) treatment. Similarly, emerging evidence has suggested that the DNA-PK complex plays a role in the cellular response to oxidative stress. We have reported that DNA-PKcs deficient cells are sensitive to H2O2 treatment and display an elevation in reactive oxygen species (ROS) production, which leads to hyper-activation of the ATM signaling pathway, including ATM, Chk2, and p53, in response to H2O2 exposure. This hyper-reactivity toward oxidative stress, however, was not observed in other NHEJ defective cells (e.g. Ligase4 knockout cells).
Our findings reveal that DNA-PKcs is required for cellular resistance to oxidative stress and suppression of ROS build-up independently of its function in DSB repair. Indeed, deficiency of DNA-PKcs also results in dysregulation of mitochondrial member potential and oxidative phosphorylation.