Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins.
Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.
Yabuuchi
Hayato
H
Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
Yamada
Yoshiki
Y
Uchida
Tomonori
T
Sunathvanichkul
Tul
T
Nakagawa
Takuro
T
Masukata
Hisao
H
D018797:Cell Cycle Proteins / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008957:Models, Genetic; D046912:Multiprotein Complexes / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D018741:Replication Origin / Q000235:genetics*; D016196:S Phase; D012568:Schizosaccharomyces / Q000166:cytology / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
C116395:CDC45 protein, S pombe; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; C496523:Sld3 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases