SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast.
Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1-S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41+ encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing.
Nakajima
Reiko
R
Department of Biology, Graduate School of Science, Osaka University, 560-0043, Japan.
Masukata
Hisao
H
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D004261:DNA Replication / Q000502:physiology; D004271:DNA, Fungal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D064151:Minichromosome Maintenance Complex Component 6; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D018741:Replication Origin / Q000502:physiology; D016196:S Phase / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D016415:Sequence Alignment
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C426020:Sld3 protein, S cerevisiae; C491207:MCM6 protein, S cerevisiae; D064151:Minichromosome Maintenance Complex Component 6