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Published on: September 11, 2022
Multiple mechanisms contribute to Schizosaccharomyces pombe origin recognition complex-DNA interactions
Christopher R Houchens1, Wenyan Lu, Ray-Yuan Chuang
1Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. chouchens@sainc.com
The Journal of Biological Chemistry
|August 30, 2008
Summary
Schizosaccharomyces pombe origin recognition complex (ORC) and its associated proteins, SpCdc18 and SpCdt1, form stable DNA replication pre-complexes through multi-step interactions. These interactions are crucial for initiating DNA replication at origins.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Eukaryotic DNA replication initiates at origins via pre-replication complexes (pre-RCs).
- Understanding the precise interactions within pre-RCs is vital for comprehending DNA replication control.
Purpose of the Study:
- To investigate the interactions between purified Schizosaccharomyces pombe pre-RC components (SpORC, SpCdc18, SpCdt1) and ars1 origin DNA.
- To elucidate the roles of these proteins in forming stable origin recognition complexes.
Main Methods:
- Purification of Schizosaccharomyces pombe pre-RC components (SpORC, SpCdc18, SpCdt1).
- Analysis of protein-DNA interactions using biochemical assays.
- Assessment of DNA topological changes upon complex formation.
Main Results:
- SpORC binds ars1 DNA in two steps: a salt-sensitive electrostatic interaction followed by a salt-stable interaction.
- Negative supercoiling facilitates SpORC binding and induces DNA topological changes, a conserved property also observed with human ORC.
- SpCdc18 and SpCdt1 form a binary complex with higher DNA affinity, stabilizing the SpORC-DNA complex and enhancing topological changes.
Conclusions:
- Stable pre-RC formation at S. pombe replication origins involves intricate binary interactions among SpORC, SpCdc18, and SpCdt1.
- SpORC alone is not the sole determinant of origin recognition; cooperative interactions are essential.
- Conserved DNA topological changes induced by ORC highlight its fundamental role in eukaryotic DNA replication initiation.
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