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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

Published: April 28th, 2021

DOI:

10.3791/62553

1Oncology Division, Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, 2Department of Molecular Biology, Medical Biochemistry, and Pathology, Laval University Cancer Research Center, 3Oncology Division, CHU de Québec Research Center, CHUL Pavilion
* These authors contributed equally

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

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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.

  1. Day 1
    1. In a 6-well plate, place a single coverslip in each well for as many conditions as needed. Plate ~150,000 HeLa cells for transfection or drug treatment, as desired.
      NOTE: If transfecting, it is recommended to do a reverse transfection at the time of plating, or it is possi.......

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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,

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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.......

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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.

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Name Company Catalog Number Comments
Alexa 568 goat anti-rabbit Molecular probes A11011 1:800
Alexa Fluor 488 goat anti-mouse Molecular probes A11001 1:800
Anti PARP1 (F1-23) Homemade 1:2500
Anti PCNA (SY12-07) Novus NBP2-67390 1:500
Anti-Alpha tubulin (DM1A) Abcam Ab7291 1:100000
anti-BrdU GE Healthcare RPN202 1:1000
Benchtop X-ray Irradiator Cell Rad
BMN673 MedChem Express HY-16106
Bromodeoxyuridine (BrdU) Sigma B5002
BSA Sigma A7906
Cell profiler Broad Institute V 3.19 https://cellprofiler.org/
Curwood Parafilm M Laboratory Wrapping Film 4in / 250 ft Fisher scientific 13-374-12
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Invitrogen Life Technology D1306
DMEM high glucose Fisher scientific 10063542
EGTA Sigma-Aldrich E3889
Fetal Bovine serum Gibco 12483-020
Fisherbrand Cover Glasses: Squares 22 x 22 Fisher scientific 12 541B
Fluorescent microscope Leica DMI6000B 63x immersion objective
HeLa ATCC CCL-2
HERACELL 160I CO2 INCUBATOR CU 1-21 TC 120V VWR 51030408 37% CO2
MgCl2 BioShop Canada MAG520.500
NaCl BioShop Canada SOD002.10
Needle
PBS 1x Wisent Bio Products 311-010-CS
PFA 16% Cedarlane Labs 15710-S(EM)
PIPES Sigma-Aldrich P6757-100G
ProLong Gold Antifade Mountant Invitrogen Life Technology P-36930
RNAiMAX Invitrogen 13778-075
siPARPi Dharmacon AAG AUA GAG CGU GAA GGC GAA dTdT
siRNA control Dharmacon UUCGAACGUGUCACGUCAA
Sodium Deoxycholcate Sigma-Aldrich D6750-100G
Sucrose BioShop Canada SUC507.5
Tris-base BioShop Canada TRS001.5
Trition X-100 Millipore Sigma T8787-250ML
Tween20 Fisher scientific BP337500
Tweezers

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