Negative regulation of Cdc18 DNA replication protein by Cdc2.
Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.
Lopez-Girona
A
A
Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Mondesert
O
O
Leatherwood
J
J
Russell
P
P
D016203:CDC2 Protein Kinase / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019926:Cyclin B; D016213:Cyclins / Q000502:physiology; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005982:Glutathione Transferase / Q000235:genetics; D051738:Origin Recognition Complex; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D018797:Cell Cycle Proteins; C080328:Cig2 protein, S pombe; D019926:Cyclin B; D016213:Cyclins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; C086077:Rum1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C082462:cdc18 protein, S pombe; C097704:orc2 protein, S pombe; D013912:Threonine; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase