Single Molecule Analysis of Laser Localized Psoralen Adducts

Summary

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

Abstract

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Introduction

DNA is under constant assault from exogenous agents such as radiation, ultraviolet light, environmental toxins, combustion products, etc. Additionally, it is also attacked by endogenous radical species produced by oxidative metabolism. All of these have the potential to chemically or physically disrupt the integrity of DNA ¹. Perturbations in the genome can activate the DNA Damage Response (DDR), a recruitment and post translational modification cascade with hundreds, if not thousands, of proteins and microRNAs involved in lesion repair, regulation of the cell cycle, apoptosis, senescence, and inflammatory pathways ².

Protocol

1. Preparation of Dig-TMP

1. Mix 50 mg (0.18 mmoles) of 4'-chloromethyl-4,5',8-trimethylpsoralen and 590 mg (2.7 mmoles) of 4,7,10-trioxa-1,13-tridecanedi-amine in a dry 25 mL round-bottom flask under nitrogen. Add 10 mL toluene and reflux for 12 h. Remove solvent in a rotary evaporator under reduced pressure.
   1. Purify the residue by flash column chromatography over silica gel. Elute the column with chloroform, methanol, and 28% ammonia solution (9:1:0.5). Evaporate the solvent in a rotary evaporator under reduced pressure, and recover the pure 4'-[N-(13-amino-4,7,10-trioxatrideca)]aminomethyl-4,5',8-trimethylpsorale....

Results

Laser localized Dig-TMP (Figure 1A) ICLs can be displayed by immunofluorescence against the Dig-tag linked to the psoralen. Although the laser can be directed to strike in an area of any contour, stripes are not "natural" shapes in cells, and legitimate signals can be easily distinguished from artifacts due to non-specific binding by primary or secondary antibodies. This feature is helpful when using antibodies of less than perfect specificity. An example of the well know.......

Discussion

The laser localization technology requires the use of adherent cells with nuclei that are visible in bright field microscopy. We have tried to attach nonadherent cells, such as primary lymphocytes, or loosely adherent cultured cells such as AD293, to the glass surface with cell adhesive preparations such as polylysine or collagen, or more complex mixtures. Although these treatments may bind the cells to the surface, we find that they generally stay rounded making it very difficult to focus into the nuclei. Furthermore, t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Digoxigenin NHS ester	Sigma-Aldrich	11333054001
Chloro-psoralen	Berry and Associates	PS 5000
diaminoglycol	Sigma-Aldrich	369519	4,7,10-Trioxa-1,13-tridecanediamine
Chloroform	Acros Organics	423550040
Methanol	Fisher Scientific	A4524
Ammonium solution	Sigma-Aldrich	5002
TLC plates	Analtech, Inc.	P02511
Flass glass column 24/40, 100 mL	Chemglass Life Sciences	CG-1196-02
Nikon T2000_E2 spinning disk confocal microscope, equipped with automated stage and environmental control chamber and plate holder	Perkin Elmer		With Volocity Software
Micropoint Galvo	Andor Technologies		with a Nitrogen pulsed laser
dye cell	Andor Technologies	MP-2250-2-365
365 dye	Andor Technologies	MP-27-365-DYE
IdU	Sigma-Aldrich	17125
35 mm glass botomm plates 1.5 coverslip, 10 mm glass diameter, uncoated	Matek	P35G-1.5-10-C
microscope slides	New Comer Supply	Part # 5070	New Silane Slides
Mouse anti BrdU antibody (IdU)	BD Biosciences	347580	1 in 40
Rat anti BrdU Antibody (CldU)	Abcam	ab6326	1 in 200
Rabbit anti Dig antibody	ThermoFisher Scientific	710019	1 in 200
Q-dot 655 goat anti Rabbit IgG	ThermoFisher Scientific	Q-11421MP	1 in 5,000
AF647- goat anti Rat IgG	Jackson Immunoresearch	112-605-167	1 in 100
AF488-goat anti mouse IgG	Jackson Immunoresearch	115-545-166	1 in 100
Zeiss epifluorescent microscope A200	Zeiss		with Axiovision software
Q-dot 655 filter	Chroma	39107