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Molecular and Cellular Biology, March 2009, p. 1134-1142, Vol. 29, No. 5
0270-7306/09/$08.00+0     doi:10.1128/MCB.00971-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Eric Weterings,1,{dagger} Nicole S. Verkaik,2,{dagger} Guido Keijzers,2,{dagger} Bogdan I. Florea,2 Shih-Ya Wang,1 Laura G. Ortega,1 Naoya Uematsu,1 David J. Chen,1* and Dik C. van Gent2*

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, Dallas, Texas 75390-9187,1 Department of Cell Biology and Genetics, Erasmus MC, University Medical Center, Dr. Molewaterplein 50, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands2

Received 19 June 2008/ Returned for modification 24 July 2008/ Accepted 2 December 2008

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-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS 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-PKCS 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-PKCS 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-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

* Corresponding author. Mailing address for David J. Chen: Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9187. Phone: (214) 648-5597. Fax: (214) 648-5995. E-mail: david.chen{at}utsouthwestern.edu. Mailing address for Dik C. van Gent: Department of Cell Biology and Genetics, Erasmus MC, University Medical Center, Dr. Molewaterplein 50, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. Phone: 31-10-4087932. Fax: 31-10-4089468. E-mail: d.vangent{at}erasmusmc.nl

{triangledown} Published ahead of print on 22 December 2008.

{dagger} E.W., N.S.V., and G.K. contributed equally to this study.

Molecular and Cellular Biology, March 2009, p. 1134-1142, Vol. 29, No. 5
0270-7306/09/$08.00+0     doi:10.1128/MCB.00971-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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