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Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
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Fluorescence Microscopy for Analysis of Relocalization of Structure-Specific Endonucleases.
Carl P Lehmann1, Irene Saugar1, José Antonio Tercero2
1Centro de Biología Molecular Severo Ochoa (CSIC/UAM), Cantoblanco, Madrid, Spain.
Methods in Molecular Biology (Clifton, N.J.)
|August 26, 2020
Summary
This study presents fluorescence microscopy protocols for analyzing protein relocalization in live budding yeast. These methods track structure-specific endonucleases, crucial for genome stability, during DNA damage and cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Protein relocalization is key to understanding genome integrity maintenance.
- Structure-specific endonucleases are vital for genome stability.
- In budding yeast, these endonucleases form subnuclear foci after DNA damage.
Purpose of the Study:
- To describe protocols for fluorescence microscopy analysis of live budding yeast cells.
- To enable the study of endonuclease relocalization during the cell cycle and DNA damage.
- To provide a framework for analyzing other protein dynamics.
Main Methods:
- Live-cell fluorescence microscopy.
- Utilizing fluorescently-tagged proteins in budding yeast.
- Analysis of protein relocalization under various cellular conditions (cell cycle, DNA damage).
Main Results:
- Established protocols for observing protein relocalization in live budding yeast.
- Demonstrated the utility of these methods for studying endonuclease dynamics.
- Showcased the applicability to cell cycle and DNA-damage responses.
Conclusions:
- The described fluorescence microscopy protocols are effective for studying protein relocalization in budding yeast.
- These methods facilitate research into genome integrity maintenance.
- The protocols are adaptable for analyzing diverse protein behaviors in live cells.
