ABSTRACT
During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.
Subject(s)
Adenosine Triphosphatases/genetics , Amphibian Proteins/genetics , Cell Cycle Proteins/genetics , DNA End-Joining Repair , Ku Autoantigen/genetics , Osteoblasts/metabolism , Recombinational DNA Repair , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amphibian Proteins/metabolism , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , Gene Expression Regulation , Humans , Hydrolysis , Ku Autoantigen/metabolism , Microspheres , Osteoblasts/cytology , Ovum/chemistry , Ovum/cytology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Valosin Containing Protein , Xenopus laevisABSTRACT
The p97-Ufd1-Npl4 ATPase complex is associated with the response to DNA damage and replication stress, but how its inactivation leads to manifestation of chromosome instability is unclear. Here, we show that p97-Ufd1-Npl4 has an additional direct role in the G2/M checkpoint. Upon DNA damage, p97-Ufd1-Npl4 binds CDC25A downstream of ubiquitination by the SCF-ßTrCP ligase and facilitates its proteasomal degradation. Depletion of Ufd1-Npl4 leads to G2/M checkpoint failure due to persistent CDC25 activity and propagation of DNA damage into mitosis with deleterious effects on chromosome segregation. Thus, p97-Ufd1-Npl4 is an integral part of G2/M checkpoint signaling and thereby suppresses chromosome instability.