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.
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Acknowledgement
ZL is the incumbent of the Maxwell Ellis Professorial Chair in Biomedical Research. This work was supported by grants to ZL from the Flight Attendant Medical Research Institute, Florida, USA, the Leona M. and Harry B. Helmsley Charitable Trust, NY, USA, and the Israel Science Foundation (no. 564/04 and 1136/08).
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Ziv, O., Diamant, N., Shachar, S., Hendel, A., Livneh, Z. (2012). Quantitative Measurement of Translesion DNA Synthesis in Mammalian Cells. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_35
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DOI: https://doi.org/10.1007/978-1-61779-998-3_35
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