Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosi s in response to DNA damage [1], This checkpoint is enforced by Chk1, a pro tein kinase that targets Cdc25 [2-7], Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS ) and thereby inhibit Cdc25 nuclear import [6, 8-14], Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fissi on yeast, We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoin t. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforceme nt. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1 , as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is s ufficient for proficient checkpoint regulation of Cdc25 and may be the prim ary mechanism of checkpoint enforcement in fission yeast.