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Molecular and Cellular Biology, October 2006, p. 7520-7528, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.00048-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress ^,

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390,¹ Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan²

Received 9 January 2006/ Returned for modification 24 February 2006/ Accepted 1 August 2006

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

Published ahead of print on 14 August 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

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