Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Summary

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

Abstract

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (γ-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Introduction

Cells are constantly exposed to endogenous and exogenous sources of DNA damage that threaten their genomic integrity. The DDR is an ensemble of signaling pathways that detect, signal, and repair DNA lesions to sustain genome stability. At DNA double-stranded breaks (DSBs), the DDR occurs mainly on two complementary platforms: γ-H2A.X-labeled chromatin and a resected single-stranded DNA (ssDNA) region typically coated with the ssDNA-binding complex RPA^1,2.

UV-laser micro-irradiation of cells pre-sensitized with the thymidine analogue 5-bromo-2'deoxyuridine (BrdU) or the bisb....

Protocol

1. Pre-sensitization of Cells

1. 24 h prior to the micro-irradiation experiment, seed 40,000 U2OS cells per well in 1x DMEM containing 10% FBS in 8-well culture slides with 170 µm-thick coverslip-like glass/polymer bottoms to ensure maximal transmittance of 405 nm laser light and excellent imaging with a laser-scanning inverted confocal microscope. If using an 8-well microslide, use 500 µL of media or solutions per well for all subsequent treatments and wash steps of the protocol.
   NOTE: Due to their flat morphology, good adherence, and large nuclei, U2OS cells facilitate the detection of protein recruitment at micro-irradiated sites but other adherent ce....

Results

Following micro-irradiation, cells are allowed to recover for a specific period of time depending on the nature of the genome maintenance proteins^1,12. DSBs will be processed by nucleases, most extensively during the S and G2 phases of the cell cycle, to create a limited region of ssDNA which is rapidly coated by RPA and other genome maintenance factors⁹. This ssDNA region is surrounded by chromatin which i.......

Discussion

Using the methods outlined above, 405 nm laser micro-irradiation of cells pre-sensitized with BBET or BrdU allows the localized generation of DNA lesions including DSBs within the nuclei of adherent human cells. Kinetics of DDR at these DSBs are similar to those generated with other methods in eukaryotic cells^9,18,19. DSB resection at micro-irradiated sites leads to ssDNA-generation which can be followed using a RPA32-targeting .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Olympus FLUOVIEW FV3000 resonant laser scanning confocal microscope	Olympus
FluoView FV3000 software (version 2.1)	Olympus
405 nm laser	Coherent		Radiation source
Microscope incubator AIO-UNO-OLYUS-1	Okolab	OKO-UNO-1
60X /1.4 Objective	Olympus		Oil immersion objective
8-well culture slides on coverglass (X-well)	Sarstedt	94.6190.802
U2OS osteosarcoma human cell line	ATCC	HTB-96	Stable cell lines
4’,6-Diamidino-2-Phenylindole (DAPI)	ThermoFisher	D1306	Caution toxic
5-bromo-2′-deoxyuridine (BrdU)	Sigma-Aldrich	59-14-3
Paraformaldehyde	Sigma-Aldrich	30525-89-4	Caution toxic
Bisbenzimide H 33342 (Hoechst 33342)	ThermoFisher	62249
Bovine serum albumin (BSA)	ThermoFisher	BP1600-100
Trypsin EDTA 0.1%	ThermoFisher	15400-054
Triton X-100	BioShop	TRX777.100
Tween-20	ThermoFisher	BP337-500
Sucrose	BioShop	SUC507
JetPRIME transfection reagent	Polyplus	114-07
ProLong Diamond Antifade mountant with DAPI	ThermoFisher	P36962
DMEM 1X, + phenol red	Wisent	319-005-CL
DMEM 1X, - phenol red	Wisent	319-051-CL
Fetal bovine serum (FBS)	Wisent	080-150
Mouse anti-RPA32 antibody	Abcam	ab2175	1:500 for IF
Rabbit anti-γ-H2A.X antibody	Cell Signaling	9718S	1:500 for IF
Rabbit Anti-53BP1 antibody	Cell Signaling	4937S	1:500 for IF
Rabbit Anti-PRP19 antibody	Abcam	ab27692	1:500 for IF
Goat anti-rabbit IgG Alexa Fluor 647	Cell Signaling	4414S	1:250 for IF
Goat anti-mouse  IgG Alexa Fluor 488	Cell Signaling	4408S	1:250 for IF
Fiji image analysis open-source software			https://fiji.sc
1918-K Power meter  with a 918D-SL-0D3 sensor	Newport