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.

ACKNOWLEDGMENTS

We thank Benjamin P. Chen for kindly providing anti-pS2056 antibody, Pierre-Olivier Mari for technical advice, and Anthony Davis and Sandeep Burma for critical revision of the manuscript.

This study was supported by NIH (grants 5-R37-CA050519-16 and PO1-CA92584), NASA (grants NNA05CM04G and NNZ07AU42G), the Association for International Cancer Research (grant 02-071), and the European Union (integrated projects RISC-RAD [contract FI6R-CT-2003-508842] and DNA Repair [contract LSHG-CT-2005-512113]).