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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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 antibody or an RPA32-GFP fusion. Chromatin-associated factors, on the other hand, can be located using &#947;-H2A.X as reference. Some chromatin-associated factors may not spread away from the initial DSB as much as &#947;-H2A.X and will appear as punctate foci that may be distinct from ssDNA-binding proteins foci.
Once DSBs are generated, protein recruitment and clearance from damage sites can be monitored by IF on fixed cells. Performing these experiments requires only a suitable IF antibody against the POI. If IF-compatible antibodies are unavailable, plasmids can be engineered carrying genes of interest in frame with specific epitopes (e.g.: FLAG, His-tag) or fluorescent proteins. Transient or stable transfection/transduction of these plasmids into adherent cells can then be used to determine whether a specific factor is actively recruited to damaged DNA. Overexpression of proteins can lead to mis-localization and may artificially boost their recruitment to micro-irradiated stripes. To obtain results that are more representative of endogenous proteins, CRISPR-Cas9 technology can now be used routinely to tag any gene of interest with suitable epitopes20,21.
The Python scripts provided in the micro-irradiation Analysis Fiji Plugin facilitate the visualization, presentation, publication, and analysis of DDR proteins kinetics. This analysis package is open-source and functions irrespective of the microscope platform used to perform micro-irradiation experiments and image acquisition. All that is required is that the images taken on the microscopy platform be saved as Bio-Formats compatible files22. Since background, drifting, and bleaching corrections are automatically performed, analysis of protein levels at DNA damage sites with the provided scripts is straightforward and very fast compared with the typical manual signal quantification performed with microscope image acquisition software.
As described in the protocol, users must carefully configure their respective laser-scanning confocal microscope systems in order to achieve the appropriate amount of damage and induce DSBs that follow normal DDR kinetics. Indeed, the quantity and type of DNA lesions generated by micro-irradiation will depend on the laser wavelength and dose as well as on the pre-sensitization methods. Users need to keep in mind that even though DSBs are generated by the conditions described here, 405 nm laser micro-irradiation of photosensitized cells will also generate a mixture of other DNA lesions, including cyclopyrimidine dimers (CPDs), SSBs, and oxidized bases, that will vary between microscope systems and settings23,24,25. Thus, a careful monitoring of the lesions at micro-irradiated sites needs to be performed to obtain a comprehensive picture of the damage generated and the response elicited. Importantly, to study the response to DSBs, each microscope system should be optimized by monitoring the kinetics of canonical DNA damage proteins and post-translational modifications at micro-irradiated sites. For instance, &#947;-H2A.X and the RPA complex subunits (RPA70, RPA32 or RPA14) can be used conveniently as they are quite easy to detect and rapidly accumulate at DSBs. As a rule of thumb, &#947;-H2A.X signal should be visualized as stripes at early (5 min) and late (up to 3&#8211;4 h) time points post-damage whereas RPA should start accumulating as punctate foci approximately 10-15 min post-micro-irradiation. If a pan-nuclear &#947;-H2A.X signal is observed, the amount of energy received by the cells is non-physiological and will result in rapid cell death.
There are some limitations that have to be considered when using a 405 nm laser for the study of specific DNA repair pathways. For example, although the BBET-405 nm combination readily produces a combination of SSBs and DSBs, it does not generate 6&#8211;4 photoproducts (6&#8211;4 PPs) and consequently, the kinetics of recruitment of nucleotide excision repair proteins are different than those observed using UV-C micro-irradiation. The kinetics and extent of recruitment of various proteins involved in the response to DSBs may also vary according to the DNA damaging method that is being used23. Because of these variations, we recommend that users adapt their systems to maximize the type of lesions that are potentially recognized by their POIs. Although such systems are less broadly available, using other types of lasers including UV-A (337&#8211;355 nm) or femtosecond near-infrared (800 nm) along with different sensitization reagents (e.g.: tri-methyl psoralen) can allow users to generate more specific DNA lesions depending on the DNA repair pathways under study24,26.
Whenever new conditions are being tested, it is convenient to use fluorescently-labeled well-established DNA repair proteins to test doses, wavelengths, and pre-sensitizer combinations, and to ensure that the lesion of interest is efficiently generated. For example, PARP1 and XRCC1 can function as markers for SSB and base-excision repair, XPA is a good indicator of nucleotide excision repair, and 53BP1 will be recruited to DSBs and function in non-homologous end-joining (references23,25,27,28). Antibodies that recognize modifications associated with specific types of DNA damage can also be used to quantify the levels of DNA adducts or breaks. It is thus possible to monitor oxidized bases, cyclopyrimidine dimers, 6&#8211;4 PPs, SSBs (using poly-ADP ribose antibody), and DSBs (&#947;-H2A.X and 53BP1) by IF and determine the parameters that will achieve the highest level of the desired lesions23,27,29. With careful monitoring, users should be able to study their repair pathway of interest using micro-irradiation.
Altogether, the use of wide-spread laser-scanning confocal microscopes equipped with 405 nm laser lines to generate localized DSBs, combined with platform-specific DNA damage markers to sub-classify genome maintenance factors, and streamlined data processing with the help of the micro-irradiation Analysis Fiji plugin, should render the study of DDR factors dynamics more accessible than ever before to both experienced and novice investigators of the genome maintenance field.

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Olympus FLUOVIEW FV3000 resonant laser scanning confocal microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:171px;"></td>
			<td style="width:179px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:508px;">FluoView FV3000 software (version 2.1)</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td>Radiation source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Microscope incubator AIO-UNO-OLYUS-1</td>
			<td>Okolab</td>
			<td>OKO-UNO-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">60X /1.4 Objective</td>
			<td>Olympus</td>
			<td></td>
			<td>Oil immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">8-well culture slides on coverglass (X-well)</td>
			<td>Sarstedt</td>
			<td>94.6190.802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">U2OS osteosarcoma human cell line</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td>Stable cell lines&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">4&#8217;,6-Diamidino-2-Phenylindole (DAPI)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>Caution toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">5-bromo-2&#8242;-deoxyuridine&#160;(BrdU)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>59-14-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td>Caution toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Bisbenzimide H 33342 (Hoechst 33342)</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Bovine serum albumin (BSA)</td>
			<td>ThermoFisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Trypsin EDTA 0.1%&#160;</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Triton X-100&#160;</td>
			<td>BioShop</td>
			<td>TRX777.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Tween-20&#160;</td>
			<td>ThermoFisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Sucrose&#160;</td>
			<td>BioShop</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">JetPRIME transfection reagent&#160;</td>
			<td>Polyplus</td>
			<td>114-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">ProLong Diamond Antifade mountant with DAPI&#160;</td>
			<td>ThermoFisher</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">DMEM 1X, + phenol red</td>
			<td>Wisent&#160;</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">DMEM 1X, - phenol red</td>
			<td>Wisent</td>
			<td>319-051-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fetal bovine serum (FBS)</td>
			<td>Wisent&#160;</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Mouse anti-RPA32 antibody</td>
			<td>Abcam</td>
			<td>ab2175</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit anti-&#947;-H2A.X antibody</td>
			<td>Cell Signaling&#160;</td>
			<td>9718S</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit Anti-53BP1 antibody</td>
			<td>Cell Signaling&#160;</td>
			<td>4937S</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Rabbit Anti-PRP19 antibody</td>
			<td>Abcam</td>
			<td>ab27692</td>
			<td>1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Goat anti-rabbit IgG Alexa Fluor 647</td>
			<td>Cell Signaling&#160;</td>
			<td>4414S</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Goat anti-mouse&#160; IgG Alexa Fluor 488&#160;</td>
			<td>Cell Signaling&#160;</td>
			<td>4408S</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fiji image analysis open-source software&#160;</td>
			<td></td>
			<td></td>
			<td>https://fiji.sc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">1918-K Power meter&#160; 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 is extensively modified during the DDR to regulate the recruitment and exchange of other DDR factors13. Protein recruitment to micro-irradiated regions can be monitored by IF (Figure 1A). Typically, chromatin-associated factors (e.g., &#947;-H2A.X, 53BP1, etc.) are detectable at earlier (1&#8211;5 min) time points than ssDNA-bound proteins because nuclease-mediated resection of DSB ends is required to generate the RPA-ssDNA platform (&#8805;10 min). To facilitate image analysis and publication, we have devised a Fiji script (The Outliner)10 which automatically outlines cell nuclei using a DNA dye-associated channel (e.g., DAPI), making it easier to distinguish micro-irradiation stripes and individual cells by leaving out uninformative DNA staining from the final images while preserving nuclear definition (Figure 1B).
&#947;-H2A.X and RPA complex subunits function as markers for chromatin- and ssDNA-associated factors, respectively. RPA-ssDNA appears as punctate foci surrounded by large fluorescent chromatin stripes decorated by the &#947;-H2A.X antibody (Figure 1C). Co-localization with these markers can be used to precisely define the position of genome maintenance factors at DNA lesions. For instance, 53BP1, a chromatin-associated protein which controls the DSB repair pathway choice, co-localizes well with the &#947;-H2A.X signal13. Conversely, PRP19, an E3 ubiquitin ligase that functions on RPA-ssDNA to promote ATR activation and DNA repair is recruited as punctate foci that closely match the RPA32 distribution14,15,16,17 (Figure 1D). Of note, some factors may also be recruited on the chromatin adjacent to the initial lesions but do not spread to large domains surrounding the DSBs like &#947;-H2A.X and 53BP1. In such cases, these factors would appear as punctate foci but would not perfectly co-localize with ssDNA-bound proteins.
For live cell imaging, cells expressing a POI tagged with a fluorescent marker can be micro-irradiated and imaged in real-time to obtain highly detailed recruitment kinetics (Figure 2A-C). Multiple different proteins can be observed simultaneously provided that their fluorescent labels can be spectrally separated from each other. To facilitate analysis of time series, we have created two Fiji scripts which allow the user to produce movie files containing time-series information (Movie Maker, see movies of Figure 2C as Online Supplementary Material) and to quantify protein recruitment at micro-irradiated sites (Damage Analyzer)10. Instead of the time-consuming manual image curation normally required to describe the behavior of a protein, the Damage Analyzer script guides the user through streamlined steps which monitor signal enrichment at micro-irradiated sites, while automatically correcting for cell movement, image drift, background signal, and bleaching at all time points of the experiment. The generated data can then be easily compiled into a spreadsheet application and plotted as needed (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57410/57410fig1v2.jpg" /> 
Figure 1: Localization of genome maintenance factors to the chromatin and RPA-ssDNA platforms at micro-irradiation-induced lesions. (A) Laser micro-irradiation in pre-sensitized cells leads to the production of DNA double-stranded breaks, which are resected by endo- and exo-nucleases to generate ssDNA. The ssDNA sub-compartment can be visualized as punctate foci using antibodies against Replication Protein A or other ssDNA-localized factors. In contrast, proteins or modifications that spread to large chromatin domains appear as large stripes at the micro-irradiated site similar to the pattern observed with antibodies targeting the phosphorylated histone variant H2A.X (&#947;-H2A.X). (B) Cells pre-sensitized with BBET or BrdU were micro-irradiated, pre-extracted, fixed, and stained for the indicated proteins. 12-bit images were collected at 1X zoom and processed using the Outliner Fiji script. (C) Z-stacking of BBET or BrdU pre-sensitized cells allows the resolution of ssDNA- and chromatin-associated factors (maximum intensity Z-projection shown). (D) Using this method, 53BP1 and PRP19 can be classified as chromatin- and ssDNA-associated genome maintenance factors, respectively. Scale bars = 10 &#181;m; images in D are all at the same scale. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57410/57410fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57410/57410fig2.jpg" /> 
Figure 2: Live-imaging of RPA complex recruitment to sites of DNA damage. (A) Stable or transient transfection of plasmids encoding candidate proteins tagged with fluorescent reporters followed by micro-irradiation of transfected cells allows the monitoring of protein recruitment to DNA lesions in real-time. (B) Cells were transfected with a pDEST47-RPA32-GFP plasmid and lysed 24 h later. Protein expression was confirmed by immunoblotting. (C) Cells transfected with pDEST-SFB EGFP or pDEST47 RPA32-GFP plasmids pre-sensitized with BrdU were micro-irradiated and imaged at 12-bit at 1X zoom once per minute post-irradiation. Timing is in minutes post-irradiation. The 00:00 time point image was taken prior to micro-irradiation. Movie files were created using the Movie Maker Fiji script and key frames are shown. Scale bars = 10 &#181;m. (D) Recruitment of RPA32-GFP to micro-irradiation stripes was quantitatively monitored using the Damage Analyzer Fiji script. Output data were plotted using Microsoft Excel. The black line is the mean enrichment-fold of RPA32-GFP at micro-irradiated sites relative to the pre-irradiation signal and the error bars represent the standard error of the mean of 15 independently micro-irradiated cells. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57410/57410fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/57410/57410mov1.jpg" /> 
Movie 1: Micro-irradiation time-lapse of SFB-GFP expressing cells. Cells transiently transfected with a pDEST-SFB GFP plasmid were pre-sensitized 24 h with BrdU, micro-irradiated, and imaged once per minute for 29 min. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57410/57410_Movie_1.avi" target="_blank">Please click here to download this movie.</a>
<img alt="Movie 2" src="/files/ftp_upload/57410/57410mov2.jpg" /> 
Movie 2: Micro-irradiation time-lapse of RPA32-GFP expressing cells. Cells transiently transfected with a pDEST47 RPA32-GFP plasmid were pre-sensitized 24 h with BrdU, micro-irradiated, and imaged once per minute for 29 min. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57410/57410_Movie_2.avi" target="_blank">Please click here to download this movie.</a>

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

Nanyang Technological University

Research

JoVE Journal

Genetics

9.3K 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, ...

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 ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

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 ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.