Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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

Protocol

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.......

Representative Results

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,

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Alexa 568 goat anti-rabbitMolecular probesA110111:800
Alexa Fluor 488 goat anti-mouseMolecular probesA110011:800
Anti PARP1 (F1-23)Homemade1:2500
Anti PCNA (SY12-07)NovusNBP2-673901:500
Anti-Alpha tubulin (DM1A)AbcamAb72911:100000
anti-BrdUGE HealthcareRPN2021:1000
Benchtop X-ray IrradiatorCell Rad
BMN673MedChem ExpressHY-16106
Bromodeoxyuridine (BrdU)SigmaB5002
BSASigmaA7906
Cell profilerBroad InstituteV 3.19https://cellprofiler.org/
Curwood Parafilm M Laboratory Wrapping Film 4in / 250 ftFisher scientific13-374-12
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)Invitrogen Life TechnologyD1306
DMEM high glucoseFisher scientific10063542
EGTASigma-AldrichE3889
Fetal Bovine serumGibco12483-020
Fisherbrand Cover Glasses: Squares 22 x 22Fisher scientific12 541B
Fluorescent microscopeLeicaDMI6000B63x immersion objective
HeLaATCCCCL-2
HERACELL 160I CO2 INCUBATOR CU 1-21 TC 120VVWR5103040837% CO2
MgCl2BioShop CanadaMAG520.500
NaClBioShop CanadaSOD002.10
Needle
PBS 1xWisent Bio Products311-010-CS
PFA 16%Cedarlane Labs15710-S(EM)
PIPESSigma-AldrichP6757-100G
ProLong Gold Antifade MountantInvitrogen Life TechnologyP-36930
RNAiMAXInvitrogen13778-075
siPARPiDharmaconAAG AUA GAG CGU GAA GGC GAA dTdT
siRNA controlDharmaconUUCGAACGUGUCACGUCAA
Sodium DeoxycholcateSigma-AldrichD6750-100G
SucroseBioShop CanadaSUC507.5
Tris-baseBioShop CanadaTRS001.5
Trition X-100Millipore SigmaT8787-250ML
Tween20Fisher scientificBP337500
Tweezers

References

  1. Mouw, K. W., Goldberg, M. S., Konstantinopoulos, P. A., D'Andrea, A. D. DNA damage and repair biomarkers of immunotherapy response. Cancer Discovery. 7 (7), 675-693 (2017).
  2. Scully, R., Panday, A., Elango, R., Willis, N. A.

Explore More Articles

DNA Double strand BreakDNA ResectionCell CycleBrdU LabelingImmunostainingImaging

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved