Related Experiment Video
Updated: Nov 7, 2025
Laser Micro-Irradiation to Study DNA Recruitment During S Phase
Published on: April 16, 2021
Laser Micro-Irradiation to Study DNA Recruitment During S Phase
Bearach Miwatani-Minter1, Gergely Rona2
1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine; Laura and Isaac Perlmutter Cancer Center, New York University School of Medicine.
Abstract:
DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.
Insights
This study details a new method to observe DNA repair dynamics specifically during the S phase of the cell cycle. It uses fluorescent microscopy to track repair proteins at DNA damage sites, offering insights into genetic integrity maintenance.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Cellular DNA damage repair is vital for maintaining genetic integrity against endogenous and exogenous threats.
- Understanding DNA repair dynamics across the cell cycle, particularly during DNA synthesis (S phase), is crucial but poorly understood.
- The S phase presents unique challenges for DNA repair due to ongoing replication and potential for new errors.
Purpose of the Study:
- To develop and present a protocol for measuring DNA repair protein dynamics in S phase cells at sites of induced DNA damage.
- To investigate the differences in DNA repair mechanisms during S phase compared to other cell cycle phases.
Main Methods:
- Utilizes laser-scanning confocal microscopy with a 405 nm laser line for localized DNA damage induction.
- Employs fluorescently tagged proteins: PCNA (Proliferating Cell Nuclear Antigen) with mPlum as an S phase marker and AcGFP-labeled repair proteins (e.g., EXO1b).
- Measures the recruitment kinetics of DNA repair proteins to damage sites within S phase cells.
Main Results:
- Successfully demonstrates a method to visualize and quantify DNA repair protein dynamics in S phase cells.
- Provides a basis for understanding how DNA repair pathways function differently during DNA replication.
- Highlights the importance of studying cell cycle-specific DNA repair.
Conclusions:
- The presented protocol enables detailed investigation of S phase DNA repair dynamics.
- This research contributes to a deeper understanding of how cells maintain genomic stability during replication.
- Further studies can build upon this method to explore various repair proteins and their roles in S phase.
