Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe.
Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Recent work has suggested that the mitotic form of cyclin-dependent kinase (Cdc2/cyclin B) has a role in preventing re-replication of the genome before mitosis, but the relevant targets of this inhibition are unknown. In this report we present evidence that the mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of Cdc18, a critical regulator of S-phase entry. We found that the ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits Cdc2 phosphorylation of histone H1 or a Cdc18 fusion protein by directly interacting with the Cdc2/cyclin B complex. Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of Cdc18 protein by a largely post-transcriptional mechanism. Overexpression of SIC1, an unrelated cyclin-dependent kinase inhibitor from budding yeast, causes a similar accumulation of Cdc18 and also leads to re-replication. Our data link a potent inhibitor of Cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggest that inhibition of Cdc18 by cyclin-dependent kinases has an important role in ensuring that the genome is duplicated precisely once each cell cycle.
Jallepalli
P V
PV
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Kelly
T J
TJ
D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004791:Enzyme Inhibitors; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D005800:Genes, Fungal; D005982:Glutathione Transferase / Q000235:genetics; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008938:Mitosis / Q000235:genetics; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D013489:Suppression, Genetic; D013831:Thiamine / Q000378:metabolism
D018797:Cell Cycle Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D006657:Histones; C093953:NMT1 protein, S pombe; D011993:Recombinant Fusion Proteins; C086077:Rum1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C082462:cdc18 protein, S pombe; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase; D013831:Thiamine