The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends

Abstract

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK CS ) and the XRCC4/ligase IV complex. Activation of the DNA-PK CS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK CS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK CS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK CS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

Identification and Functional Characterization of a Ku-binding Motif in Aprataxin Polynucleotide Kinase/Phosphatase-like Factor (APLF)

“…The C-terminal Region of Ku80 Is Not Required for Interactions with APLF-Although Ku80 is highly homologous to Ku70, the C-terminal region of Ku80 is divergent and has been shown to be required for cellular radioresistance (30). This region of Ku80 is also required for DNA-PKcs autophosphorylation and activation of Artemis end processing (30).…”

“…This region of Ku80 is also required for DNA-PKcs autophosphorylation and activation of Artemis end processing (30). Therefore, we questioned whether this C-terminal portion of Ku80 might be important for directing interactions with APLF.…”

The DNA-dependent protein kinase (DNA-PK): More than just a case of making ends meet?

“…38,39 Furthermore, Ku80 has been shown to stimulate joining and Artemismediated processing of DNA ends. 40 The most widely investigated system of NHEJ is V(D)J recombination that is responsible for antibody diversity and normal immune development (reviewed in ref. 41 and 42).…”

Dynamics of the PI3K-like protein kinase members ATM and DNA-PKcs at DNA double strand breaks

“…Similar to this, further studies with DNA-PKcs and Ku80 have shown that the Ku80 C-terminal motif is not essential for DNA-PKcs kinase activity or its ability to localize to DSBs. 38 However, deletion of the C-terminal portion of Ku80 results in radiosensitivity, which is probably due to its importance for phosphorylation of DNA-PKcs at the 2609 cluster and the DNA end processing by the Artemis endonuclease and ultimately end-joining. These data suggest that ATM and DNA-PKcs are both recruited to DSBs via multimeric complexes, but further detailed studies need to be performed to delineate the specific domains/residues required for localization of ATM and DNA-PKcs to DSBs.…”