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Updated: May 19, 2026
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
Quantitative measurement of translesion DNA synthesis in mammalian cells
Omer Ziv1, Noam Diamant, Sigal Shachar
1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism, in which specialized low-fidelity DNA polymerases bypass lesions that interfere with replication. This process is inherently mutagenic due to the miscoding nature of DNA lesions, but it prevents double strand breaks, genome instability, and cancer. We describe here a quantitative method for measuring TLS in mammalian cells, based on non-replicating plasmids that carry a defined and site-specific DNA lesion in a single-stranded DNA region opposite a gap. The assay is responsive to the cellular composition of TLS DNA polymerases, and TLS regulators. It can be used with a broad variety of cultured mammalian cells, and is amenable to RNAi gene silencing, making it a useful tool in the study of TLS in mammalian cells.
Insights
A new quantitative method measures translesion DNA synthesis (TLS) in mammalian cells. This assay helps study DNA repair polymerases and regulators, preventing genome instability and cancer.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- Translesion DNA synthesis (TLS) is crucial for bypassing DNA lesions during replication.
- While mutagenic, TLS prevents catastrophic DNA damage, genome instability, and cancer.
- Specialized low-fidelity DNA polymerases execute TLS.
Purpose of the Study:
- To develop a quantitative method for measuring TLS in mammalian cells.
- To provide a tool for studying TLS polymerases and regulatory factors.
- To enable research into preventing DNA damage-induced mutations and cancer.
Main Methods:
- Utilized non-replicating plasmids with site-specific DNA lesions in single-stranded regions.
- Designed the assay to be quantitative and responsive to cellular TLS machinery.
- Applied RNA interference (RNAi) gene silencing for functional studies.
Main Results:
- Developed a robust assay for quantifying TLS in various cultured mammalian cells.
- Demonstrated the assay's responsiveness to cellular TLS polymerase composition and regulators.
- Validated the method's utility in conjunction with RNAi for gene silencing studies.
Conclusions:
- The developed quantitative TLS assay is a valuable tool for mammalian cell research.
- This method facilitates the study of DNA damage tolerance mechanisms.
- Understanding TLS is critical for insights into genome stability and cancer prevention.
