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MBC in Press, published online ahead of print June 11, 2004
Mol. Biol. Cell 10.1091/mbc.E04-04-0292

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Submitted on April 8, 2004
Revised on June 1, 2004
Accepted on June 3, 2004

A Novel Intermediate in Initiation Complex Assembly for Fission Yeast DNA Replication

Yoshiki Yamada, Takuro Nakagawa, and Hisao Masukata*

Graduate School of Science, Osaka University, Osaka 560-0043 Japan

Monitoring Editor: Keith Yamamoto

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of pre-initiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41+/cdc45+. The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably due to a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.

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




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