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Molecular and Cellular Biology, August 2006, p. 6056-6064, Vol. 26, No. 16
0270-7306/06/$08.00+0     doi:10.1128/MCB.00492-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Claspin Operates Downstream of TopBP1 To Direct ATR Signaling towards Chk1 Activation{ddagger}

Shizhou Liu,1,2,{dagger} Simon Bekker-Jensen,1,{dagger} Niels Mailand,1 Claudia Lukas,1 Jiri Bartek,1 and Jiri Lukas1*

Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark,1 Department of Medical Oncology, The First Affiliated Hospital, China Medical University, Shenyang 110001, People’s Republic of China2

Received 21 March 2006/ Returned for modification 28 April 2006/ Accepted 2 June 2006

TopBP1 and Claspin are adaptor proteins that facilitate phosphorylation of Chk1 by the ATR kinase in response to genotoxic stress. Despite their established requirement for Chk1 activation, the exact way in which TopBP1 and Claspin control Chk1 phosphorylation remains unclear. We show that TopBP1 tightly colocalizes with ATR in distinct nuclear subcompartments generated by DNA damage. Although depletion of TopBP1 by RNA interference (RNAi) strongly impaired phosphorylation of multiple ATR targets, including Chk1, Nbs1, Smc1, and H2AX, it did not interfere with ATR assembly at the sites of DNA damage. These findings challenge the current concept of ATR activation by recruitment to damaged DNA. In contrast, Claspin, like Chk1, remained distributed throughout the nucleus both before and after DNA damage. Consistently, the RNAi-mediated ablation of Claspin selectively abrogated ATR's ability to phosphorylate Chk1 but not other ATR targets. In addition, downregulation of Claspin mimicked Chk1 inactivation by inducing spontaneous DNA damage. Finally, we show that TopBP1 is required for the DNA damage-induced interaction between Claspin and Chk1. Together, these results suggest that while TopBP1 is a general regulator of ATR, Claspin operates downstream of TopBP1 to selectively regulate the Chk1-controlled branch of the genotoxic stress response.

* Corresponding author. Mailing address: Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Phone: 45 35 25 73 10. Fax: 45 35 25 77 21. E-mail: jil{at}cancer.dk.

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

{dagger} These authors contributed equally to the paper.

Molecular and Cellular Biology, August 2006, p. 6056-6064, Vol. 26, No. 16
0270-7306/06/$08.00+0     doi:10.1128/MCB.00492-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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