TYROSINE PHOSPHORYLATION OF CDC2 IS REQUIRED FOR THE REPLICATION CHECKPOINT IN SCHIZOSACCHAROMYCES-POMBE

The DNA replication checkpoint inhibits mitosis in cells that are unab le to replicate their DNA, as when nucleotide biosynthesis is inhibite d by hydroxyurea. In the fission yeast Schizosaccharomyces pombe, gene tic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contr ary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylatio n of Cdc2 on tyrosine-15 is not part of the replication checkpoint mec hanism. We have undertaken biochemical and genetic studies to resolve this controversy. We report that the DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressin g an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc 25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be main tained at a low level that is insufficient to induce mitosis. These st udies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is esse ntial for the DNA replication checkpoint and suggests that Cdc25, and/ or one or both of Wee1 and Mik1, the tyrosine kinases that phosphoryla te Cdc2, are regulated by the replication checkpoint.