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Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level
Published on: July 26, 2024
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A helicase-tethered ORC flip enables bidirectional helicase loading
Shalini Gupta1, Larry J Friedman2, Jeff Gelles2
1Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, United States.
Elife
|December 9, 2021
Summary
A single origin recognition complex (ORC) molecule loads two Mcm2-7 helicases for DNA replication. ORC rearranges its interaction with the first helicase, enabling the loading of the second, ensuring proper replication origin licensing.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA replication initiation requires loading of two Mcm2-7 helicases at origins by ORC, Cdc6, and Cdt1.
- The precise stoichiometry of ORC in helicase loading has remained an open question.
Purpose of the Study:
- To investigate the role of ORC in recruiting Mcm2-7 helicases during origin licensing.
- To elucidate the dynamic interactions between ORC and Mcm2-7 during the loading of two helicases.
Main Methods:
- Utilized colocalization single-molecule spectroscopy.
- Employed single-molecule Förster resonance energy transfer (FRET) to study ORC-Mcm2-7 interactions.
Main Results:
- Observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes.
- Identified a rapid rearrangement of ORC interaction with the first Mcm2-7 after its recruitment.
- Demonstrated ORC flipping over the first Mcm2-7 to form an inverted complex essential for second helicase loading.
Conclusions:
- A single ORC molecule is sufficient for loading two Mcm2-7 helicases in a head-to-head conformation.
- ORC undergoes a dynamic conformational change, involving release and re-binding, to facilitate the loading of the second helicase.
- This coordinated mechanism ensures the precise licensing of DNA replication origins.
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