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Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples
Published on: May 5, 2017
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MCM double hexamer loading visualized with human proteins.
Florian Weissmann1, Julia F Greiwe2, Thomas Pühringer2
1Chromosome Replication Laboratory, The Francis Crick Institute, London, UK.
Nature
|November 28, 2024
Summary
Human DNA replication initiation involves loading the MCM helicase. This study reveals distinct human double hexamer loading mechanisms compared to yeast, highlighting differences in protein interactions and assembly pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Eukaryotic DNA replication initiates with MCM helicase loading as a double hexamer at replication origins.
- Current models for double hexamer assembly by ORC, CDC6, and CDT1 are primarily based on budding yeast.
Purpose of the Study:
- To biochemically reconstitute and structurally characterize human double hexamer (hDH) loading.
- To elucidate the mechanistic differences in hDH assembly compared to the yeast double hexamer (yDH).
Main Methods:
- Biochemical reconstitution using purified human proteins.
- Cryo-electron microscopy (cryo-EM) for structural analysis.
- Comparative analysis with existing yeast data.
Main Results:
- The hDH engages DNA differently than yDH, creating underwound DNA at the hexamer interface.
- Identified distinct factor recruitment orders and dependencies during hDH assembly.
- ORC6 is not essential for initial MCM recruitment but facilitates an alternative pathway involving ORC1's disordered region.
Conclusions:
- Human double hexamer assembly differs significantly from yeast, indicating diversity in eukaryotic replication initiation.
- This work provides a detailed view of hDH assembly in an organism with sequence-independent origins.
- Represents a foundational step towards reconstituting human DNA replication initiation in vitro.
