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A novel intermediate in initiation complex assembly for fission yeast DNA replication
Yoshiki Yamada1, Takuro Nakagawa, Hisao Masukata
1Graduate School of Science, Osaka University, Osaka 560-0043 Japan.
Molecular Biology of the Cell
|June 15, 2004
Summary
Replication initiation requires MCM and Cdc45 loading. Fission yeast studies reveal Sld3 loading precedes Cdc45 loading, forming a key preinitiation complex intermediate for DNA replication.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- DNA replication initiation is tightly regulated at the S phase onset.
- Assembly of preinitiation complexes at replication origins is crucial for controlling replication timing and S-phase checkpoint responses.
Purpose of the Study:
- To investigate the ordered assembly of replication initiation factors at origins in fission yeast.
- To understand the roles of MCM and Cdc45 in preinitiation complex formation using a specific fission yeast mutant.
Main Methods:
- Utilized a temperature-sensitive mcm5 mutant in fission yeast (nda4-108/mcm5).
- Examined the genetic interactions between mcm5 and cdc45.
- Analyzed the loading of MCM, Cdc45, and Sld3 proteins onto replication origins under different temperature conditions.
Main Results:
- The mcm5 mutation impaired Cdc45 loading but not MCM loading onto origins.
- Sld3 proficiently associated with origins in the mcm5 mutant, independent of Cdc45.
- Origin association of Sld3 without Cdc45 was also observed in a cdc45 mutant.
- Restoring permissive temperature to arrested mcm5 cells led to immediate Cdc45 loading and DNA replication resumption.
Conclusions:
- Sld3 loading onto origins is independent of Cdc45 loading in fission yeast, differing from budding yeast.
- A complex containing MCM and Sld3 represents a crucial intermediate in the initiation of DNA replication in fission yeast.