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Molecular and Cellular Biology, May 2001, p. 3398-3404, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3398-3404.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Threonine-11, Phosphorylated by Rad3 and ATM In Vitro, Is Required for Activation of Fission Yeast Checkpoint Kinase Cds1

Katsunori Tanaka, Michael N. Boddy, Xiao-Bo Chen, Clare H. McGowan, and Paul Russell*

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Received 23 October 2000/Returned for modification 4 December 2000/Accepted 20 February 2001

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T11Q12. Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1- null mutant. Cds1T11A was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

* Corresponding author. Mailing address: Department of Molecular Biology, MB3, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-2823. Fax: (858) 784-2265. E-mail: prussell{at}scripps.edu.

Molecular and Cellular Biology, May 2001, p. 3398-3404, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3398-3404.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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