Published: April 28th, 2021
In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.
The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2).
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2′-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.
Modulation of DNA repair factors is an ever-evolving method for cancer therapy, particularly in DNA DSB repair-deficient tumor environments. The inhibition of specific repair factors is one of the ingenious strategies used to sensitize cancer cells to DNA-damaging agents. Decades of research led to the identification of various mutations of DNA repair genes as biomarkers for therapeutic strategy choices1. Consequently, the DNA repair field has become a hub for drug development to ensure a wide range of treatments, empowering the personalized medicine concept.
DSBs are repaired by two main pathways: NHEJ and HR
1. Cell culture, treatments, and coverslip preparation
NOTE: All cell plating, transfections, and treatments, aside from irradiation, should take place under a sterile cell-culture hood.
In this protocol, the bromodeoxyuridine (BrdU)-based assay was used to quantitatively measure the resection response of HeLa cells to irradiation-induced damage. The generated ssDNA tracks are visualized as distinct foci after immunofluorescence staining (Figure 1A). The identified foci were then quantified and expressed as the total integrated intensity of the BrdU staining in the nuclei (Figure 1B, Supplemental Figure S1, Supplemental Figure S2,
This paper describes a method that makes use of IF staining to measure variations in DNA resection in cellulo. The current standard for observing an effect on DNA resection is through RPA staining; however, this is an indirect method that may be influenced by DNA replication. Previously, another BrdU incorporation-based DNA resection IF technique has been described for classifying the resulting intensities in BrdU-positive and BrdU-negative cells. This method allowed for cells that are not undergoing HR to be co.......
We thank Marie-Christine Caron for outstanding technical advice. This work is supported by funding from Canadian Institutes of Health Research J.Y.M (CIHR FDN-388879). J.-Y.M. holds a Tier 1 Canada Research Chair in DNA Repair and Cancer Therapeutics. J.O'S is an FRQS PhD student fellow, and S.Y.M is a FRQS postdoctoral fellow.....
|Alexa 568 goat anti-rabbit
|Alexa Fluor 488 goat anti-mouse
|Anti PARP1 (F1-23)
|Anti PCNA (SY12-07)
|Anti-Alpha tubulin (DM1A)
|Benchtop X-ray Irradiator
|Curwood Parafilm M Laboratory Wrapping Film 4in / 250 ft
|DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)
|Invitrogen Life Technology
|DMEM high glucose
|Fetal Bovine serum
|Fisherbrand Cover Glasses: Squares 22 x 22
|63x immersion objective
|HERACELL 160I CO2 INCUBATOR CU 1-21 TC 120V
|Wisent Bio Products
|ProLong Gold Antifade Mountant
|Invitrogen Life Technology
|AAG AUA GAG CGU GAA GGC GAA dTdT
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