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Observing protein dynamics during DNA-lesion bypass by the replisome
Elise M Wilkinson1,2, Lisanne M Spenkelink1,2, Antoine M van Oijen1,2
1Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia.
Frontiers in Molecular Biosciences
|October 10, 2022
Summary
Understanding DNA replication dynamics is key to preventing diseases like cancer. This review explores ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy to study replisome-lesion interactions and protein dynamics during DNA repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Faithful DNA replication is crucial for life, ensuring genomic stability and preventing diseases such as cancer.
- Replication roadblocks, like DNA damage from UV light, challenge DNA replication fidelity, leading to genomic instability and increased mutational load.
- Protein dynamics are critical for molecular pathways involved in DNA damage bypass and replication restart.
Purpose of the Study:
- To review and discuss three key methods for studying protein dynamics during replisome-lesion encounters.
- To highlight the application of these methods in model DNA replication systems from Escherichia coli and Saccharomyces cerevisiae.
Main Methods:
- Ensemble biochemical assays: Provide indirect insights into protein dynamics through averaged measurements.
- Single-molecule fluorescence microscopy: Enables real-time visualization of individual protein dynamics during replisome-lesion interactions.
- Cryo-electron microscopy (including time-resolved variants): Offers high-resolution structural information and visualizes dynamic processes.
Main Results:
- Ensemble assays have been historically important but lack direct dynamic readouts.
- Single-molecule techniques allow direct observation of specific protein movements during DNA replication and damage encounters.
- Cryo-electron microscopy provides structural context, with time-resolved methods bridging the gap between static structures and dynamics.
Conclusions:
- A combination of ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy provides a powerful toolkit for studying DNA replication dynamics.
- These integrated approaches are essential for visualizing the complex interplay between the replisome, DNA lesions, and protein dynamics in real time.
- Understanding these dynamics is vital for deciphering mechanisms of DNA repair and developing strategies to combat diseases linked to replication errors.
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