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

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 possible to do a forward transfection several hours after plating to allow for adherence. A reverse transfection is accomplished by adding the transfection....

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.......

Materials

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