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MBC in Press, published online ahead of print February 28, 2002
Mol. Biol. Cell 10.1091/mbc.02-01-0006

A more recent version of this article appeared on May 1, 2002
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Submitted on November 18, 2001
Revised on January 7, 2002
Accepted on January 24, 2002

SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast

Reiko Nakajima1 and Hisao Masukata1*

1 Department of Biology, Graduate School of Science, Osaka University 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan

* Corresponding author. E-mail address: masukata{at}bio.sci.osaka-u.ac.jp.

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 kinase (DDK) 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 pre-loaded Cdc45 from chromatin in the hydroxyurea (HU)-arrested S phase, and DNA replication upon removal of HU 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.




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