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Updated: Jun 17, 2025
08:53
Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
300
Dynamics of Replication-Associated Protein Levels through the Cell Cycle.
Aleksandar Atemin1, Aneliya Ivanova1, Petar-Bogomil Kanev1
1Laboratory of Genomic Stability, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G., Bonchev Str. Bl. 21, 1113 Sofia, Bulgaria.
International Journal of Molecular Sciences
|August 10, 2024
Summary
This study introduces a live-cell imaging method to track protein dynamics during the cell cycle, offering high spatiotemporal resolution for replication factors. This approach enhances our understanding of cell cycle-coordinated processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Understanding dynamic protein changes during the cell cycle is crucial for studying replication, transcription, DNA repair, and checkpoint control.
- Existing methods for measuring protein levels lack the necessary temporal and spatial resolution.
- Live-cell imaging offers a potential solution for observing protein dynamics with greater precision.
Purpose of the Study:
- To develop and validate a live-cell imaging approach for measuring dynamic changes in protein levels and localization throughout the cell cycle.
- To achieve high spatiotemporal resolution in observing cell cycle-associated protein dynamics.
- To compare the cell cycle-dependent behavior of multiple replication-associated factors.
Main Methods:
- Utilized genetic engineering and live-cell microscopy for time-lapse imaging of fluorescently tagged proteins.
- Employed proliferating cell nuclear antigen (PCNA)-mCherry to distinguish cell cycle phases and subphases based on fluorescence.
- Quantified levels and distribution of replication factors like RIF1, MCM6, ORC1, and Claspin in HeLa Kyoto cells.
Main Results:
- Successfully discerned cell cycle phases and S subphases using PCNA-mCherry fluorescence intensity and distribution.
- Precisely determined and compared the dynamic levels and localization of RIF1, MCM6, ORC1, and Claspin.
- Integrated imaging data with mass spectrometry to reveal protein concentration changes throughout the cell cycle.
Conclusions:
- The developed live-cell imaging method provides high spatiotemporal resolution for interrogating protein dynamics during the cell cycle.
- This approach enables detailed analysis of replication-associated protein behavior in coordination with cell cycle progression.
- Offers a practical foundation for future studies on cell cycle regulation and associated molecular events.
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