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. 2023 Sep 15;4(3):102361.
doi: 10.1016/j.xpro.2023.102361. Epub 2023 Jun 23.

Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells

Tom Egger  1 , Antoine Aze  1 , Domenico Maiorano  2
Affiliations

Protocol to analyze endogenous translesion DNA synthesis in single mammalian cells

Tom Egger et al. STAR Protoc. .

Abstract

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.

Keywords: Cancer; Cell Biology; Molecular Biology.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Representative bright field (100×) images of either non pre-extracted (top) or pre-extracted (bottom) HEK293T cells in PBS 0,2% Triton-X100 for 3min on ice
Figure 2
Figure 2
Pre-gating general workflow performed on Kaluza Acquisition Software for flow cytometry analysis (related to 7. b. II-V)
Figure 3
Figure 3
Typical localization of endogenous PCNAmUb bound to chromatin by immunofluorescence (left panel) in either untreated cells (NT) or upon exposure to 20 J/m2 and its detection by flow cytometry (right panel) in pre-extracted HCT116 cells
See this image and copyright information in PMC

References

    1. Egger T., Aze A., Maiorano D. Detection of endogenous translesion DNA synthesis in single mammalian cells. Cell Rep. 2023 doi: 10.1016/j.crmeth.2023.100501. - DOI - PMC - PubMed
    1. Forment J.V., Walker R.V., Jackson S.P. A high-throughput, flow cytometry-based method to quantify DNA-end resection in mammalian cells. Cytometry. 2012;81:922–928. doi: 10.1002/cyto.a.22155. - DOI - PMC - PubMed
    1. Stirling D.R., Swain-Bowden M.J., Lucas A.M., Carpenter A.E., Cimini B.A., Goodman A. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinf. 2021;22:433. doi: 10.1186/s12859-021-04344-9. - DOI - PMC - PubMed

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