Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells
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Published on: December 22, 2023
Tom Egger1, Antoine Aze1, Domenico Maiorano1
1Institut de Génétique Humaine (IGH) CNRS UMR9002, Université de Montpellier, Department, Molecular Bases of Human Diseases, "Genome Surveillance and Stability" Laboratory, 34396 Cedex 5, Montpellier, France.
Abstract:
Translesion DNA synthesis (TLS) is an evolutionarily conserved branch of the cellular DNA damage tolerance pathway that is often exploited by cancer cells to overcome therapy resistance. Here, we present a protocol to analyze endogenous TLS in single mammalian cells in the absence or presence of DNA damage. We describe steps for detecting chromatin-bound TLS factors, such as monoubiquitinated PCNA(mUb) and TLS DNA polymerases (pols) by flow cytometry. We then detail a procedure to detect their nuclear localization using immunofluorescence. For complete details on the use and execution of this protocol, please refer to Egger et al. (Cell Reports Methods, in press).1.
Insights
This study introduces a new protocol to analyze translesion DNA synthesis (TLS) in single cells. The method detects key TLS factors, aiding cancer therapy resistance research.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Translesion DNA synthesis (TLS) is a crucial DNA damage tolerance pathway.
- Cancer cells utilize TLS to evade therapeutic DNA damage, leading to resistance.
- Analyzing endogenous TLS in single cells is vital for understanding this process.
Purpose of the Study:
- To present a novel protocol for analyzing endogenous translesion DNA synthesis (TLS) in single mammalian cells.
- To enable the detection of TLS factors in both normal and DNA-damaged cellular conditions.
- To provide a method for assessing TLS activity relevant to cancer therapy resistance.
Main Methods:
- Flow cytometry is used to detect chromatin-bound TLS factors, including monoubiquitinated PCNA (mUb).
- TLS DNA polymerases (pols) are also quantified using flow cytometry.
- Immunofluorescence is employed to determine the nuclear localization of these TLS factors.
Main Results:
- The protocol successfully allows for the detection of endogenous TLS factors in single cells.
- It differentiates between the presence or absence of DNA damage.
- The method facilitates the analysis of chromatin association and nuclear localization of key TLS proteins.
Conclusions:
- This protocol offers a robust method for studying translesion DNA synthesis at the single-cell level.
- It provides insights into TLS factor dynamics relevant to DNA repair and cancer progression.
- The technique is valuable for research into overcoming therapy resistance in cancer.
