Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cancer
• Molecular Biology