PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

Abstract

The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N’ terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

DNA–dependent protein kinase in telomere maintenance and protection

“…2). This is consistent with our findings that DNA-PKcs is critically involved in the cellular response to replication stress, and it coordinates with the ATR signaling pathway for optimal replication checkpoint and fork recovery [75,81,111].…”

“…It is possible that Ku and DNA-PKcs contribute to the telomeric capping and maturation process in distinct ways. For example, DNA-PKcs is required to resolve the stalled replication fork in telomeres and participates in replication stress signaling independently of Ku [75,76].…”

The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage

“…It is unclear how our respective studies could come to such different conclusions at this point, so further replication studies will be required in future. Furthermore, it has been suggested that DNA-PKcs also plays roles in responses to stalled replication forks besides DSB repair 38,39 . Our mass spectrometry and ensuing immunoprecipitation analysis indicated that UCHL3 could interact with DNA-PKcs, possibly suggesting context specific functions of UCHL3 for maintaining genome integrity.…”

Protective Mechanisms Against DNA Replication Stress in the Nervous System

“…In the context of replication stress, it was shown that ATR phosphorylates DNA-PK following UV-induced replication stress and that the DNA-PK function facilitates DNA damage resolutions [310]. The phosphorylation of DNA-PK can reinforce the ATR-Chk1-mediated DDR by promoting a claspin-Chk1 interaction stability, and it was proposed that DNA-PK may act as a signal amplifier of ATR [311,312]. Moreover, upon ATR inhibition, DNA-PK can act as a backup pathway that phosphorylates Chk1 and other targets, suppressing origin firing [299].…”