Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast

Genome integrity is monitored by a checkpoint that delays mitosis in respon se to DNA damage. This checkpoint is enforced by Chk1, a protein kinase tha t inhibits the mitotic inducer Cdc25. In fission yeast, Chk1 is regulated b y a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR. These kinases phosphorylate serine or threonine followed by g lutamine (SQ/TQ). Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367. Serine-345 of human Chk1 is phosph orylated in response to DNA damage. Here we report that Rad3 and ATM phosph orylate serine-345 of fission yeast Chk1. Mutation of serine-345 (chk1-S345 A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo. The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective. In contras t, mutations of serine-367 and other SQ/TQ sites do not substantially impai r the checkpoint or cause damage sensitivity. These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen e vidence that transduction of the DNA damage checkpoint signal requires dire ct phosphorylation of Chk1 by Rad3.