Threonine-11, phosphorylated by Rad3 and ATM in vitro, is required for activation of fission yeast checkpoint kinase Cds1

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage, Cds1 enforces the S-M c heckpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regul ated by a group Of proteins that includes Rad3, a kinase related to human c heckpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates se rine or threonine followed by glutamine (SQ or TQ). Here we show that in vi tro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12). Substitution Of threonine-ll with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (H U) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant. Cds1(T11A) was unable to enforce tile S-M checkpoint. These results strongly suggest that Rad3-depen dent phosphorylation of Cds1 at threonine-ll is required for Cds1 activatio n and function.