Name: investigation of protein recruitment to dna lesions using 405 nm laser micro-irradiation
Uploaded: 2018-03-20T15:00:00.000+00:00
Duration: 12 min 29 s
Description: Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

This work was supported by the Natural Sciences and Engineering Research Council of Canada [Discovery grant 5026 to A.M] and by a Next-Generation of Scientists Scholarship from the Cancer Research Society [21531 to A.M]. We thank Jean-Fran&#231;ois Lucier for providing excellent quality control of the Fiji Python scripts and advice on statistical analysis. We thank Dr. Stephanie A. Yazinski for the IF fixation solution recipe. We thank Paul-Ludovic Karsenti for help with laser power measurements.

1. Pre-sensitization of Cells
<ol>
	<li>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 &#181;m-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 &#181;L 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 cell types can be used as needed. Cells should ideally be around 80% confluency at the time of micro-irradiation to increase the number of irradiated cells per experiment. Very high confluence may result in cell cycle alterations that can perturb specific repair pathways such as homologous recombination which takes place during the S and G2 phases of the cell cycle9.</li>
	<li>Incubate the cells overnight at 37 &#176;C with 5% CO2, and then pre-sensitize the cells with (step 1.3) BrdU or (step 1.4) BBET (Hoechst 33342). 
	NOTE: To observe live recruitment of a protein of interest (POI) to DNA lesions, transfect or transduce plasmid DNA carrying a fusion between a fluorescent protein and a POI 24 h after cell seeding. 24 h post-transfection/transduction, pre-sensitize and micro-irradiate the cells to monitor the recruitment of the POI at DNA damage sites in real time.</li>
	<li>To increase the number of DSBs generated by exposition to the 405 nm laser, add 10 &#181;M of BrdU to the media for 24 h prior to micro-irradiation. Prior to micro-irradiation, using a micropipette, rinse the cells twice with medium without phenol red to ensure maximal exposure of the cells to the 405 nm laser.</li>
	<li>Alternatively, treat cells for 15 min with BBET at 10 &#181;g/mL in the appropriate medium in a cell-culture incubator at 37 &#176;C. After treatment, rinse twice with medium without phenol red prior to micro-irradiation.</li>
</ol>
2. Micro-irradiation of Cells
<ol>
	<li>Select a suitable laser-scanning confocal microscope. 
	NOTE: The experiments presented here were performed on a microscope system equipped with a 50 mW 405 nm laser line and a 63X 1.4 numerical aperture oil objective. To generate DSBs, cells were micro-irradiated at 1X scan zoom at 1,024 x 1,024 pixels/field (0.21 &#181;m/pixel at 63X magnification) and in unidirectional mode at 8 &#181;s/pixel. The laser power used was 25% and 80% for BBET- and BrdU-pre-sensitized cells, respectively. To help users configure their system, the laser power post-objective on the presented system was measured using a power meter according to manufacturer&#39;s instructions. The measurements correspond to 2.6 mW and 7.0 mW for BBET- and BrdU-pre-sensitized cells, respectively.</li>
	<li>Turn on the microscope and the environmental chamber.
	<ol>
		<li>Set the chamber to 37 &#176;C with 5% CO2 before samples are placed into the on-stage incubator to avoid unnecessary stress to the cells and to ensure homogeneous DDR kinetics across experiments.</li>
	</ol>
	</li>
	<li>Select field(s) with well-distributed cells, adjust the focus, and register their position to facilitate image acquisition after the IF protocol. Acquire at least one image that will serve as a pre-damage reference point to monitor the kinetics of protein recruitment after micro-irradiation. 
	NOTE: Gridded culture vessels may also be used to facilitate the localization of micro-irradiated cells at subsequent steps.
	<ol>
		<li>For IF experiments using BBET-labeled cells, adjust the focus on cells using the 405 nm laser at the lowest laser power sufficient to visualize the cells in order to avoid unwarranted DNA damage.
		<ol>
			<li>Avoid using widefield fluorescence to adjust the focus on BBET-stained cells as the UV light emitted by the lamp will induce DNA damage at illuminated sites.</li>
		</ol>
		</li>
		<li>If using BrdU-labeled cells, adjust the focus using differential interference contrast (DIC) or phase-contrast imaging by looking at sub-nuclear features such as nucleoli.</li>
		<li>If using cells expressing fluorescently-labeled POIs, select a focal plane with representative fluorescence intensity. 
		NOTE: Avoid cells that express very high levels of the POI as strong overexpression may result in artifactual localization. Additionally, select cells that express the POI at comparable levels (selection of stable clones is strongly recommended to minimize variations in POI expression and recruitment levels).</li>
	</ol>
	</li>
	<li>Configure the microscope for the micro-irradiation step using the fluorescence-recovery after photobleaching (FRAP)_module of the system. 
	NOTE: The most important parameters to consider when setting-up the microscope system for micro-irradiation experiments are: the output power of the 405 nm laser, number of iterations (i.e., number of times the laser will go over the same coordinates), number of pixels/field (at 63X magnification and 1,024 x 1,024 resolution, 1 pixel = 0.21 &#181;m), and dwell time per pixel. All of these factors will impact the amount of energy that the cells experience and thus the amount and type of DNA damage generated (see Discussion for further dose optimization information).</li>
	<li>Using the FRAP module of the microscope system, micro-irradiate the cells. 
	NOTE: On most systems, micro-irradiation of multiple cells can be performed in a single field as spots or lines/rectangles (typically 1-2 &#181;m wide). When performing live-imaging of fluorescently-labeled proteins immediately post-damage, limit the micro-irradiation to a small number of cells per field because the time taken to micro-irradiate each cell will result in a delayed induction of the DDR. This is particularly important when monitoring proteins that are recruited very rapidly to DNA lesions.</li>
	<li>After micro-irradiation, put back the multi-well slides or plates in an incubator at 37 &#176;C with 5% CO2 for the required duration prior to processing samples for IF (Section 5) or proceed immediately with live-imaging (Section 3).
	<ol>
		<li>As a first step, fix the cells within a few minutes and up to a few hours post-irradiation for the IF protocol. Depending on the POI, recruitment kinetics may vary. Typically, proteins that are chromatin-associated at DSBs and SSBs will localize very rapidly to the lesion and are sometimes removed within 5 min. Some factors such as those that assemble at ssDNA will be recruited at later time points once the resection machinery has been engaged and remain there for hours (See Figure 1 and reference4).</li>
	</ol>
	</li>
</ol>
3. Live-imaging of Fluorescently-labeled Proteins
<ol>
	<li>Perform time-lapse imaging of the micro-irradiated cells. For proteins that are recruited very rapidly to lesions, a few seconds per frame will be ideal, but for proteins that localize to the damaged area later (e.g., resection factors), acquisition may be performed every 1&#8211;2 min until a plateau is achieved. 
	NOTE: Minimize the laser power for image acquisition to avoid bleaching as much as possible and to ensure that protein recruitment at damage sites does not reach saturation which would preclude signal quantification and subsequent analyses (see data analysis Section 4). On the presented system, the imaging was done with a 63X/1.4 oil objective using an 8 &#181;s/pixel dwell time, 1,024 x 1,024 resolution, 1X zoom, and averaging of 2. Note that these parameters may need to be optimized for other systems and/or different experiments.</li>
	<li>Micro-irradiate the cells in additional fields until kinetics measurements are obtained for 15-30 cells. Repeat the experiments at least 3 times in biological replicates to ensure the accuracy and reproducibility of the recruitment kinetics. 
	NOTE : When adapting this protocol to a specific confocal system, it may be useful to start by monitoring the recruitment kinetics of a known fluorescently-labeled genome maintenance factor (e.g., 53BP1-GFP, RPA32-GFP, etc.). Using live-imaging to optimize micro-irradiation conditions will allow the user to test multiple parameters rapidly.</li>
</ol>
4. Recruitment Kinetics Analysis
<ol>
	<li>Install the Microirradiation Analysis plugin10 in the Fiji image analysis software11.</li>
	<li>Open the acquired images in Fiji. Select &#34;The Damage Analyzer&#34; from the pull-down plugins menu in the Microirradiation Analysis suite. Follow the prompts to define a background region of interest (ROI) (typically 3 x 3 &#181;m), and non-irradiated and irradiated ROIs in each micro-irradiated cell.
	<ol>
		<li>Use ROIs of the same size to minimize potential biases in mean signal intensities. For acquired images, also include at least one pre-irradiation frame which will be used as a baseline to monitor recruitment at micro-irradiated sites.</li>
		<li>Once all ROI data have been collected over the full time-lapse series, save the file created by the plugin containing the results as a csv (comma delimited file) or txt file (tab delimited file). This file contains measurements of the mean intensity for each time point and each ROI (Mean Intensity Background, Mean Intensity Non-irradiated, and Mean Intensity Irradiated). 
		NOTE: The plugin automatically subtracts the background intensity (Ib) from the intensity of the irradiated ROIs (Is) and the non-irradiated ROIs (In). It also calculates a ratio between both values (see the formula below) to correct for photobleaching (Irradiated/Non-irradiated). 
		<img alt="Equation 1" src="/files/ftp_upload/57410/57410eq1v2.jpg" /> 
		The plugin also calculates a normalization between the first time point (value set to 1) and all the other time points for each nucleus (ratio relative to first time point). Additional information can be found on the plugin website10.</li>
	</ol>
	</li>
	<li>Perform the analysis using the plugin for all micro-irradiated cells (at least 15&#8211;30 cells) and graph the mean relative enrichment at micro-irradiated regions over time. Calculate the standard errors of the mean for each data point and plot them.
	<ol>
		<li>Confirm differences in recruitment kinetics between two experimental conditions by performing Student&#39;s t-tests for each data point.</li>
	</ol>
	</li>
</ol>
5. IF Protocol
<ol>
	<li>Prepare IF solutions. Prepare pre/post-extraction solution (ice-cold 1x PBS containing 0.25% Triton X-100), washing solution (1x PBS containing 0.05% Tween-20), blocking solution (1x PBS containing 3% BSA and 0.05% Tween-20), and fixation solution (Paraformaldehyde 3% + sucrose 2% in PBS 1x) in conical polypropylene tubes. 
	NOTE: For a single 8-well microslide, 10 mL of pre/post-extraction solution, 50 mL of washing solution, 10 mL of blocking solution, and 5 mL of fixation solution are required.
	<ol>
		<li>Prepare the fixation solution.
		<ol>
			<li>Under a chemical hood, mix 400 mL of double-distilled water (ddH2O) with 15 g of paraformaldehyde and 30 &#181;L of 10 N NaOH in a 1 L Erlenmeyer flask.</li>
			<li>Close the container and heat at 65 &#176;C on a magnetic stirrer hot plate while stirring to solubilize the paraformaldehyde completely.</li>
			<li>Using a 25-mL pipette, add 50 mL of 10X PBS, stir, and filter using a 0.45 &#181;m filter.</li>
			<li>Add 10 g of sucrose and complete to 500 mL with ddH2O.</li>
			<li>Aliquot the solution in 15 mL conical tubes; store at -20 &#176;C until use.</li>
		</ol>
		</li>
		<li>Prepare the nuclei staining solution by diluting a stock solution of 1 mg/mL of 4,6-Diamidino-2-phenylindole (DAPI) 1:1,000 in 1x PBS.</li>
	</ol>
	</li>
	<li>Using a micropipette, carefully remove the media from the wells of the multi-well microscopy slides or plates and immediately wash with 500 &#181;L of IF washing solution by changing the solution twice without waiting. 
	NOTE: The IF data presented here were obtained using cells grown in 8-well microscopy slides that were washed and incubated in 500 &#181;L of all solutions. Volumes should be adjusted depending on the cell culture containers used by experimenters. Additionally, perform the IF protocol with extreme care as cells can easily detach from the glass/polymer bottom. Use a micropipette for all solution changes to minimize cell loss.</li>
	<li>Incubate on ice for 5 min with 500 &#181;L of ice-cold IF pre/post-extraction solution and swirl gently by hand for 15&#8211;30 s to perform the pre-extraction step that removes most cytoplasmic and nucleoplasmic proteins and maximizes the signal from chromatin/DNA-associated factors.</li>
	<li>Wash twice with 500 &#181;L of IF washing solution.</li>
	<li>Incubate for 15 min at room temperature in 500 &#181;L of IF fixation solution. Alternatively, use a pre-made paraformaldehyde solution (3&#8211;4%) for the fixation step.</li>
	<li>Wash twice with 500 &#181;L of IF washing solution.</li>
	<li>Incubate on ice for 5 min in 500 &#181;L of ice-cold IF pre/post-extraction solution and swirl the slide gently by hand to perform the permeabilization step.</li>
	<li>Wash twice with 500 &#181;L of IF washing solution.</li>
	<li>Incubate at least 30 min at room temperature with 500 &#181;L of IF blocking solution.</li>
	<li>Remove blocking solution using a micropipette and incubate overnight in a humidified chamber at 4 &#176;C with 250 &#181;L primary antibodies diluted in blocking solution. 
	NOTE: It is good practice to perform the incubation with primary antibodies sequentially to avoid potential artifacts of co-localization. Once appropriate controls have been performed, simultaneously incubate primary antibodies to speed up the process.</li>
	<li>Wash four times for 5 min in 500 &#181;L of IF washing solution with gentle agitation (60 rpms on a rotator).</li>
	<li>Incubate for 1 h at 37 &#176;C with 250 &#181;L of secondary antibodies diluted in blocking solution in a humidified chamber. 
	NOTE: Protect the samples from light upon addition of fluorescently-labeled secondary antibodies.</li>
	<li>Wash four times each for 5 min in 500 &#181;L of IF washing solution with gentle agitation.</li>
	<li>Incubate at room temperature for 5 min in 500 &#181;L of 1x PBS containing 1 &#181;g/mL DAPI to stain nuclei.</li>
	<li>Wash twice with 500 &#181;L of 1x PBS and leave cells in 500 &#181;L of 1x PBS solution for imaging.</li>
	<li>Using gridded slides or registered stage positions, find the micro-irradiated cells using a well-characterized DNA damage marker (e.g., &#947;-H2A.X, RPA32, etc.).</li>
	<li>Image the multi-well culture slides or plates in all relevant channels using the appropriate settings on a laser-scanning confocal microscope. 
	NOTE: On the presented system, the imaging was done with a 63X/1.4 oil objective using an 8 &#181;s/pixel dwell time, 1,024 x 1,024 resolution, 1X zoom, and averaging of 2. Note that these parameters may need to be optimized for other systems and/or different experiments.</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+detection+of+8-oxoguanine+in+nuclear+and+mitochondrial+DNA+of+cultured+cells+using+a+recombinant+Fab+and+confocal+scanning+laser+microscopy.">Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.</a> Free Radical Biology and Medicine. 28 (6), 987-998 (2000).</li></ol>

The authors have nothing to disclose.

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 cells9,18,19. DSB resection at micro-irradiated sites leads to ssDNA-generation which can be followed using a RPA32-targeting ...

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: &#947;-H2A.X-labeled chromatin and a resected single-stranded DNA (ssDNA) region typically coated with the ssDNA-binding complex RPA1,2.
UV-laser micro-irradiation of cells pre-sensitized with the thymidine analogue 5-bromo-2&#39;deoxyuridine (BrdU) or the bisbenzimide ethoxide trihydrochloride (BBET, Hoechst 33342) DNA dye creates a mixture of DNA lesions including single-stranded breaks (SSBs) and DSBs which elicit a localized DDR on both the chromatin and ssDNA platforms3,4. Previous work showed that recruitment of genome maintenance factors to these two distinct platforms at DSB can be discriminated using high energy UV-A lasers (335-365 nm) combined with IF2,5. Microscopes equipped with such lasers are costly as they require a high energy laser and dedicated UV-A transmitting objectives making them far less prevalent in academic and pharmaceutical settings than laser-scanning confocal microscopes with 405 nm laser lines. The study of protein recruitment and exchange at micro-irradiated sites is also precluded by the laborious manual image analysis required to describe the dynamic behavior of genome maintenance factors.
Here, we show that the pre-sensitization of cells with BrdU or BBET nucleic acid stain followed by micro-irradiation using a 405 nm laser on a common confocal microscope allows the monitoring of genome maintenance factor dynamics at DNA lesions. Using &#947;-H2A.X or RPA complex subunits as platform markers together with z-stacking for greater depth of field and deconvolution for improved resolution allows the experimenter to discriminate factors that are locally recruited to DSBs from those that spread to large chromatin domains surrounding the initial lesion. This sub-classification according to distinct intra-nuclear compartments helps refine the potential roles of uncharacterized proteins recruited to micro-irradiated sites. Moreover, we provide convenient protocols and optimized pipelines to rapidly analyze the dynamics of genome maintenance factors using the open-source software Fiji (an ImageJ distribution)6,7,8. These refinements to current micro-irradiation methods render the study of the DDR possible in virtually any laboratory setting.

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

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.

Following micro-irradiation, cells are allowed to recover for a specific period of time depending on the nature of the genome maintenance proteins1,12. 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...

Watch this Scientific Journal Video about Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation at JoVE.com

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

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 ...

investigation-protein-recruitment-to-dna-lesions-using-405-nm-laser

Research

JoVE Journal

Genetics

9.4K Views.  Université de Sherbrooke. 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.

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

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

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.

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 ...

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

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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 ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Biology

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

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

Summary

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Chapters in this video

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

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

Protocols for C-Brick DNA Standard Assembly Using Cpf1

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

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

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Laser Micro-Irradiation to Study DNA Recruitment During S Phase