Name: single molecule analysis of laser localized psoralen adducts
Uploaded: 2017-04-20T12:30:00.000+00:00
Duration: 11 min 46 s
Description: Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (Z01 AG000746-08) and the Fanconi Anemia Research Fund.

1. Preparation of Dig-TMP
<ol>
	<li>Mix 50 mg (0.18 mmoles) of 4&#39;-chloromethyl-4,5&#39;,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.
	<ol>
		<li>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&#39;-[N-(13-amino-4,7,10-trioxatrideca)]aminomethyl-4,5&#39;,8-trimethylpsoralen as a pale yellow viscous liquid.</li>
		<li>Mix 5.5 mg (0.012 mmoles) of 4&#39;-[N-(13-amino-4,7,10-trioxatrideca)]aminomethyl-4,5&#39;,8-trimethylpsoralen with 5 mg (0.008 mmoles) of digoxigenin NHS ester in a dry round-bottom flask under nitrogen. Add 0.5 mL anhydrous dimethyl formamide (DMF) and 3.4 &#181;L of trimethylamine and stir at 50 &#176;C for 18 h.</li>
		<li>Remove solvent in a rotary evaporator under reduced pressure.</li>
		<li>Dissolve the residue in minimum amount of dichloromethane. Apply the solution in 2 &#181;L spots horizontally across the bottom of a preparative thin layer plate with silica gel as a stationary phase. Run the preparative TLC in chloroform: methanol: 28% ammonium hydroxide (8:1:0.1).</li>
		<li>Mask the plate except for the vertical edges and identify the band of product with short wavelength UV 254 nm lamp. Scrape the product from the plate with a spatula and isolate the pure product using chloroform: methanol: 28% ammonium hydroxide (8:1:0.1) mixture.</li>
		<li>Remove solvents in a rotary evaporator under reduced pressure. Dissolve the residual pellets in 1 mL of 50% EtOH:H2O, dispense into 50 &#181;L aliquots in 1.5 mL tubes (~20 aliquots) and dry in a centrifugal evaporator until all solvent is evaporated (~3 h).</li>
		<li>Redissolve each aliquot in 200 &#181;L of 50% EtOH:H2O and dry again in a centrifugal evaporator. Dissolve each aliquot again in 200 &#181;L of 50% EtOH:H2O, dry in a centrifugal evaporator and store pellets at -20 &#176;C for long term storage.</li>
	</ol>
	</li>
	<li>For use, dissolve the pellet in 50 &#181;L of 50% EtOH:H2O. Prepare a 1:100 dilution of the dissolved Dig-TMP in H2O and measure OD at 250 nm. The extinction coefficient of Dig-TMP is 25,000. The solution is stable for about a month if stored at -20 &#176;C. Verify the concentration by measuring OD at 250 nm before each use.
	<ol>
		<li>Calculate stock concentration: Abs x 100 x106/25,000 = Concentration (in &#181;M). Typically the stock solution is about 3 mM. It is important to be in this range in order to minimize the volume of EtOH added to the medium.</li>
	</ol>
	</li>
</ol>
2. Laser Localized Dig-TMP ICLs
<ol>
	<li>Perform laser localization with a confocal microscope with a SRS nitrogen laser (337 nm) pumped through a dye cell emitting a line of 365 nm and firing 3 ns pulses at 10 Hz, with a power of 0.7 nW.</li>
	<li>Make a vertical mark on the side of a 35 mm glass bottomed cell culture dish with a marking pen. Scratch a cross in the center of the glass on the culture surface with a diamond pen such that the black stripe on the side of the dish is at the top of one arm of the cross. The cross is marked on the growth surface of the glass so that it and the cells will be in the same focal plane.</li>
	<li>Sterilize the plate after marking with a rinse of 70% EtOH. Dry before plating cells.</li>
	<li>Plate cells (standard laboratory strains such as HeLa or U2OS) in the cross marked culture dishes one or two days before. Cell should be actively dividing and 50-70% confluent on the day of the experiment. Incubate 24 h with 1 &#181;M 5-chloro-2-deoxyuridine (CldU) to uniformly label DNA.</li>
	<li>Add Dig-TMP in 50% EtOH: H2O to cell culture medium to a final concentration of 20 &#181;M. Bring the medium to 37 &#176;C. Change the medium over the cells and place in incubator (37 &#176;C, 5% CO2) for 30 min to allow the Dig-TMP to equilibrate.</li>
	<li>While the cells are incubating, establish the x/y coordinates of the field of view in which the laser is active. Follow the manufacturer&#39;s calibration procedure in which the laser is directed by the software to etch a mirrored glass slide along a horizontal and diagonal line. The two lines define the boundaries of the field in which the laser can strike a target.</li>
	<li>Place the plate in the environmental chamber, at 37 &#176;C with controlled CO2 and humidity, on the microscope stage and focus with the 60X oil objective on the intersection of the cross.</li>
	<li>Direct the 365 nm laser beam to an ROI, established by the investigator with the shape tool in the software, to form a stripe of 3 &#181;m x 0.6 &#181;m. The outline of the ROI is placed by the cursor in nuclei of cells located in the immediate vicinity of the intersection of the cross. Adjust the z focal plane to target the laser midway through the nucleus. Use a laser in the range of 350-370 nm. 405 nm lasers cannot induce crosslinks 19. 
	NOTE: We locate the ROI in nucleoplasmic areas outside the nucleoli, which can be distinguished from nucleoplasm in differential interference contrast (DIC) imaging.</li>
	<li>Verify the focus and activity of the laser by increasing the power setting to a level sufficient to obliterate a cell (the cell will darken and then disappear). This is an important diagnostic for an unimpeded light path for the laser through the microscope and then through the coverslip and into the cells.
	<ol>
		<li>Lower the power setting to just enough to activate the psoralen and the ICL induced DDR (this must be determined empirically for each laser/microscope combination). Use a laser intensity setting (1.7% here) that does not activate the DDR in the absence of psoralen (monitored by the formation of &#947;-H2AX). 
		NOTE: This is an important determination and the settings may require adjustment if the laser source or a component in the light path is changed. Follow the accumulation of GFP tagged repair proteins such as XPC or FAN1, both of which are rapidly recruited (in seconds) to the laser psoralen stripes, but not to ROI exposed to the laser alone. Some proteins of the DDR appear within seconds, while several minutes may be required for others. It is necessary to perform time course determinations for each protein of interest. We also note that in cells that were not exposed to the laser there are no stripes of responding proteins.</li>
	</ol>
	</li>
	<li>After all cells in a field have been targeted move the plate to a new field, staying close to the intersection of the cross.</li>
	<li>In experiments designed to determine the distribution of inter lesion distances expose cells to the laser in 20-25 fields (4-5 cells/field) around the intersection. In order to analyze replication patterns in the vicinity of the localized ICLs incubate cells with 10 &#181;M 5-Iodo-2-deoxyuridine (IdU) after laser firing. 
	NOTE: The time of incubation with IdU is somewhat arbitrary. We have used as short as 20 min and as long as 60 min (see below). Longer pulses (a few hours) often result in multiple replication tracts coalescing, thus losing the opportunity to image the progress of single forks. In some experiments ces are labeled with CldU for 24 h to uniformly label DNA prior to exposure to Dig-TMP followed by pulse labeling of replication tracts.</li>
</ol>
3. Harvest of Cells and Stretching of DNA Fibers
<ol>
	<li>Remove the plate from the stage, remove the medium, and wash with phosphate buffered saline (PBS). Remove the PBS solution. Place a 10 &#181;L drop of a commercial trypsin/EDTA solution on the intersection of the cross in the middle of the glass surface.</li>
	<li>Incubate for 3-4 min at room temperature (RT) and then draw the trypsin solution with the detached cells into the pipet tip. There is no need to neutralize the trypsin.</li>
	<li>Place the drop of trypsin/EDTA with the cells at one end of a silanized glass microscope slide. Lyse the cells and release the DNA by addition of 10 &#181;L of 0.5% SDS solution and incubate for 3-4 min at RT mixing gently with the pipet tip, allowing the periphery of the pool to dry.</li>
	<li>Tilt the slide 20&#186; and allow the liquid to run down to the end. The DNA fibers are extended/stretched by the hydrodynamic forces of the flowing liquid. Let the slides air dry (~10 min) and fix in 3:1 methanol/acetic acid for 10 min. Remove the slides from the fix solution and air dry again. At this point they can be stored indefinitely in 70% EtOH at -20 &#176;C. On removal from the 70 % EtOH allow to air dry before the next step.</li>
	<li>Wash slides in PBS, and then denature in 2.5 M HCl for 1 h at RT. This treatment depurinates DNA making the incorporated halogenated nucleobases accessible to antibodies.</li>
	<li>Neutralize slides in 0.4 M Tris-HCl, pH 7.4. Wash twice in PBS/0.5% Tween-20 (PBST) for 5 min each.</li>
	<li>Block slides with 5% bovine serum albumin (BSA) and 10% goat serum in PBS. Cover the slides gently with a parafilm to spread the blocking solution evenly over the slide and incubate for 1 h at RT.</li>
	<li>Pipet 100 &#181;L of 1:200 dilution in blocking solution of rat anti-bromodeoxyuridine (specifically detects CldU) primary antibody to each slide. Cover the slides gently with a parafilm to spread the dilution evenly over the slide and incubate in a humidified chamber for 1 h at RT. Remove the parafilm and wash the slides three times in PBST for 5 min each.</li>
	<li>Pipet 100 &#181;L of diluted (1:100) goat anti-rat Alexa Fluor 647 in blocking solution to each slide. Cover the slides gently with a parafilm and incubate for 45 min at RT. Wash the slides three times in PBST for 5 min each.</li>
	<li>Pipet 100 &#181;L of diluted (1:40) mouse anti-bromodeoxyuridine (detects ldU and CldU. This dual specificity necessitates the blocking of CldU by the rat anti BrdU in the prior incubation) primary antibody and rabbit anti-DIG antibody (1:200) in blocking solution to each slide. Cover the slides gently with a parafilm and incubate in a humidified chamber for 1 h at RT. Wash the slides three times in PBST for 5 min each.</li>
	<li>Pipet 100 &#181;L of diluted (1:100) goat anti-mouse Alexa Fluor 488 andc(1:5,000) goat anti-rabbit probe (e.g., Qdot 655) in blocking solution to each slide. Cover the slides gently with parafilm and incubate for 45 min at RT. Wash the slides three times in PBST for 5 min each.</li>
	<li>Drain excess PBST onto a paper towel. Add 50 &#181;L of antifade mounting medium onto each slide and cover with a coverslip. Image or store for no more than 48 h at -20 &#176;C.</li>
</ol>
4. Fiber Imaging by Fluorescence Microscopy
<ol>
	<li>Perform image acquisition through a 63X objective on an inverted microscope with an attached fully motorized filter wheel with FITC, Cy5 and Q Dot 655 detection. The excitation filter is 425 nm, and the emission filter is 655 nm with 20 nm centered at the emission peak 20.</li>
</ol>

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The authors have nothing to disclose.

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...

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.
Most of our information about the DDR comes from studies with DSBs. This is in large part due to the availability of technologies for introducing breaks, including sequence specific breaks, in genomic DNA in living cells 3. In addition, the propensity of breaks to induce foci of DDR proteins, which can be displayed by immunofluorescence, has been very helpful for identifying the kinetics and requirements of responding proteins. One of the key technologies for studying the DDR was introduced by Bonner and colleagues, who used a laser beam to direct a stripe of DSBs in a &#34;Region of Interest&#34; (ROI) in the nuclei of living cells 4. In effect, they created a lengthy focus in which proteins of the DDR could be identified by immunofluorescence. This was illustrated by their demonstration of the strong stripe of phosphorylated histone H2AX (&#947;-H2AX) in the laser exposed cells. Since then, the laser approach has been employed in numerous studies of the DDR induced by DSBs. Although powerful and popular, and the source of dramatic immunofluorescence images, it should be noted that in most experiments the laser intensity is adjusted so as to produce observable results, without concern for lesion identity, density, or spacing. Indeed, it can be difficult to make these estimates. Thus they are largely ignored, despite the multiplicity of lesions introduced into DNA by lasers 5. This contributes to the many contradictions in the literature 6.
In contrast to DSBs, most chemical modifications of DNA do not stimulate the formation of discrete foci of DDR proteins. This is important in the light of our current understanding of lesion frequencies. It has been estimated that human cells in culture incur as many as 50 DSBs per cell cycle, formed largely during S phase 7,8,9. Fewer are formed in non-proliferating cells. This contrasts with the number of nucleobase losses or modification events, which are in the tens of thousands per cell/day 1,10. Thus, we know most about the DDR induced by events that are relatively rare, and much less about those induced by helix distorting lesions, which in aggregate are far more common.
In order to address questions about the cellular response to covalent modifications of genomic DNA, we wanted to work with a helix distorting DNA adduct that had inherent DDR induction activity. Furthermore, to facilitate experimental design and interpretation we were interested in a structure whose introduction could be controlled with respect to time and was amenable to visualization. Accordingly, we developed a strategy based on psoralen. Psoralens are well characterized photoactive DNA intercalators favoring 5&#39; TA:AT sites. Unlike other crosslinking agents such as nitrogen mustards and mitomycin C (MMC) they are not DNA reactive unless exposed to long wave UV (UVA) light. The intercalated molecules react with thymine bases on opposite strands to produce helix distorting interstrand crosslinks (ICLs) 11. With the trimethyl psoralen used in our experiments most products are ICLs, relatively few monoadducts are generated (less than 10%) 12, and intrastrand crosslinks between adjacent bases on one strand are not formed. Because they are powerful blocks to replication and transcription, psoralen and other crosslinking agents, like cis-platinum and MMC, are commonly used in chemotherapy. Thus psoralen enabled studies that followed the activation of the DDR by a helix distorting structure, and also provided insight into the cellular response to a compound with clinical importance.
We synthesized a reagent in which trimethyl psoralen was linked to digoxigenin (Dig), a plant sterol not found in mammalian cells and frequently used as an immunotag. The requirement for photoactivation permits localization by laser light (365 nm) of psoralen ICLs in defined ROI in nuclei in living cells. These can be displayed by immunofluorescence against the Dig tag. DNA repair and DDR proteins appeared in the stripes of laser localized ICLs 13,14.
The DDR activated by the high laser intensities used to produce DSBs could be due to isolated or clustered damage 15,16. Consequently, the relevance of results from these experiments to naturally occurring lesions, present at much lower concentration, is uncertain. To address similar questions about psoralen adduct frequency and spacing in DNA, we took advantage of DNA fiber technology 17 and immunoquantum dots. Quantum dots are much brighter than fluorescent dyes and are not bleached by exposure to light. Thus they are frequently used for single molecule imaging 18, an application for which fluorescent dyes are insufficiently bright. Individual DNA fibers can be stretched on glass slides and can be displayed by immunofluorescence against nucleoside analogues incorporated during incubations prior to cell harvest. We treated cells with Dig-psoralen and exposed the ROI to laser micro irradiation. Fibers were prepared from the cells and individual Dig-psoralen adducts could be visualized with the immunoquantum dots. Exposing the cells to nucleoside analogues for relatively short times (20-60 min) permits the display of replication tracts in the vicinity of the laser localized ICLs.

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

single molecule analysis of laser localized psoralen adducts

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.

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 &#34;natural&#34; 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 w...

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Single Molecule Analysis of Laser Localized Psoralen Adducts

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.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

single-molecule-analysis-of-laser-localized-psoralen-adducts

Research

JoVE Journal

Genetics

6.6K Views.  National Institute on Aging, National Institutes of Health.  Lasers worden vaak gebruikt in studies van de cellulaire respons op DNA-schade. Echter, ze genereren lesies waarvan de afstand, frequentie en botsingen met replicatievorken worden zelden gekenmerkt. We beschrijven een benadering die de bepaling van deze parameters laser gelokaliseerde crosslinks tussen de strengen mogelijk maakt. 

Video: Single Molecule Analysis of Laser Localized Psoralen Adducts

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Cancer Research

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

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

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 (&#947;-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.

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.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Biochemistry

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (&#9642;OH) to irreversibly label a protein&#8217;s solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of &#9642;OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes ...

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Immunology and Infection

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

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