Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells

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 [Display omitted] •Detection of endogenous TLS factors by flow cytometry and immunofluorescence•Monitoring chromatin-bound TLS factors in respect to cell cycle phases•Monitoring TLS factors in respect to DNA synthesis and DNA lesions Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.