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Published on: July 26, 2024
Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA
Jingchuan Sun1, Cecile Evrin, Stefan A Samel
1Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA.
Abstract:
In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.
Insights
Researchers captured a key intermediate in DNA replication initiation, the OCCM complex, revealing how the ORC-Cdc6 helicase loader interacts with MCM2-7. This structure suggests a conserved mechanism for DNA replication clamp loaders.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- DNA replication initiation is crucial for cell division in eukaryotes.
- The replicative helicase MCM2-7 is loaded onto DNA by the ORC-Cdc6 complex to form the prereplicative complex (pre-RC).
- Understanding the structural intermediates of pre-RC assembly is vital for comprehending DNA replication regulation.
Purpose of the Study:
- To capture and characterize an intermediate in prereplicative complex assembly.
- To elucidate the structural basis of ORC-Cdc6 interaction with Cdt1-MCM2-7 during DNA replication initiation.
- To investigate the mechanism of helicase loading by the ORC-Cdc6 complex.
Main Methods:
- In vitro biochemical reconstitution using purified protein components.
- Cryo-electron microscopy (Cryo-EM) for high-resolution structural analysis.
- Biophysical techniques to study complex formation and stability.
Main Results:
- Successfully captured and determined the structure of the OCCM intermediate, a 14-subunit complex of ORC-Cdc6 and Cdt1-MCM2-7.
- Revealed extensive interactions between ORC-Cdc6 AAA+ domains and MCM2-7 motor domains.
- Observed a conformational change in ORC-Cdc6 into a right-handed spiral mimicking DNA helical symmetry.
Conclusions:
- The captured OCCM structure provides unprecedented insight into the mechanism of MCM2-7 loading onto DNA.
- The structural similarity of ORC-Cdc6 to replication factor C suggests a conserved clamp-loading mechanism.
- This study advances our understanding of the fundamental process of DNA replication initiation in eukaryotes.
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