Detection of endogenous translesion DNA synthesis in single mammalian cells

Tom Egger; Antoine Aze; Domenico Maiorano

doi:10.1016/j.crmeth.2023.100501

Detection of endogenous translesion DNA synthesis in single mammalian cells

Cell Rep Methods. 2023 Jun 14;3(6):100501. doi: 10.1016/j.crmeth.2023.100501. eCollection 2023 Jun 26.

Authors

Tom Egger¹, Antoine Aze¹, Domenico Maiorano¹

Affiliation

¹ Institut de Génétique Humaine (IGH) CNRS UMR9002, Université de Montpellier, Molecular Bases of Human Pathologies Department, "Genome Surveillance and Stability" Laboratory, 34396 Cedex 5 Montpellier, France.

Abstract

Translesion DNA synthesis (TLS) is an evolutionarily conserved process that cells activate to tolerate DNA damage. TLS facilitates proliferation under DNA damage conditions and is exploited by cancer cells to gain therapy resistance. It has been so far challenging to analyze endogenous TLS factors such as PCNA^mUb and TLS DNA polymerases in single mammalian cells due to a lack of suitable detection tools. We have adapted a flow cytometry-based quantitative method allowing detection of endogenous, chromatin-bound TLS factors in single mammalian cells, either untreated or exposed to DNA-damaging agents. This high-throughput procedure is quantitative, accurate, and allows unbiased analysis of TLS factors' recruitment to chromatin, as well as occurrence of DNA lesions with respect to the cell cycle. We also demonstrate detection of endogenous TLS factors by immunofluorescence microscopy and provide insights into TLS dynamics upon DNA replication forks stalled by UV-C-induced DNA damage.

Keywords: CPDs; DNA damage tolerance; PCNAmUb; TLS pols; UV-C; XP-V; chromatin; flow cytometry; oxidative stress; replication stress.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Chromatin / genetics
DNA / genetics
DNA Repair*
DNA Replication* / genetics
DNA-Directed DNA Polymerase / genetics
Mammals / genetics

Substances

DNA-Directed DNA Polymerase
DNA
Chromatin