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Updated: Jun 5, 2026
Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG
Published on: July 26, 2024
Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload
Marjorie A Kuipers1, Timothy J Stasevich, Takayo Sasaki
1Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
Abstract:
The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.
Insights
Minichromosome maintenance (Mcm2-7) proteins load onto DNA during G1 phase and remain stably bound until replication occurs. This study shows Mcm proteins are irreversibly locked onto chromatin, ensuring stable DNA replication throughout the cell cycle.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- The minichromosome maintenance (Mcm2-7) complex is essential for eukaryotic DNA replication, functioning as the replicative helicase.
- Mcm2-7 complexes are loaded onto chromatin in early G1 phase but remain inactive until S phase.
- It is presumed that Mcm2-7 remains stably bound to chromatin throughout G1 and S phases.
Purpose of the Study:
- To investigate the in vivo dynamics of Mcm protein loading and stability on chromatin in live mammalian cells.
- To understand the mechanism retaining Mcm proteins on chromatin from G1 through S phase.
Main Methods:
- Live-cell imaging in mammalian cells.
- Analysis of Mcm protein dynamics and chromatin binding.
- Investigating Mcm protein behavior during replication arrest.
Main Results:
- Mcm proteins are irreversibly loaded onto chromatin throughout G1 phase with no detectable exchange with soluble pools.
- Mcm eviction from chromatin requires ongoing DNA replication; Mcm remains bound indefinitely upon replication arrest.
- Mcm density decreases and Mcm proteins become mobile within actively replicating chromatin regions, explaining their lack of colocalization with replication fork proteins.
Conclusions:
- Mcm proteins are retained on chromatin via an exceptionally stable "lockdown" mechanism throughout the cell cycle.
- This mechanism ensures the continuous presence of Mcm complexes for DNA replication, even during prolonged cell cycles or replication stress.
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