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Molecular and Cellular Biology, May 2007, p. 3378-3389, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.00863-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mechanisms of Checkpoint Kinase Rad53 Inactivation after a Double-Strand Break in Saccharomyces cerevisiae

Ghislaine Guillemain,^1,, Emilie Ma,^1, Sarah Mauger,¹ Simona Miron,² Robert Thai,¹ Raphaël Guérois,¹ Françoise Ochsenbein,¹ and Marie-Claude Marsolier-Kergoat^1*

CEA, Direction des Sciences du Vivant, Institut de Biologie et de Technologies de Saclay, 91191 Gif-sur-Yvette Cedex, France,¹ Institut Curie, INSERM U759, 91405 Orsay, France²

Received 15 May 2006/ Returned for modification 5 July 2006/ Accepted 9 February 2007

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G₂/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1 and ckb2 mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.

Published ahead of print on 26 February 2007.

G.G. and E.M. contributed equally to this work.

Present address: Faculté de Médecine Necker, INSERM E363, 75730 Paris, France.

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