The overall goal of this procedure is to create localized DNA double-stranded breaks using a broadly available 405 nanometer laser scanning confocal microscope, and provide automated procedures to quantify the dynamics of DNA repair factors at these lesions. This method can be used to determine whether a given protein is recruited to double-stranded DNA breaks, and delineate the signaling pathways that regulate its accumulation at DNA lesions. The technique uses a commonly available confocal microscope system to locally induce DNA damage.
This allows virtually any laboratory to study the kinetics of the DNA damage response. If monitoring the live recruitment of a fluorescently-labeled protein, perform transient transfection, or selection of stable cell lines expressing the protein of interest, fused to a fluorescent reporter. 24 hours prior to the micro-irradiation experiment, seed 40, 000 U2OS cells per well in DMEM 1X containing 10%FBS, in eight-well culture slides, with 170 micron thick coverslip-like glass polymer bottoms.
Incubate the cells overnight at 37 degrees Celsius, with 5%CO2. To increase the number of double-stranded breaks generated by exposure to the 405 nanometer laser, add BBET to the cells, at 10 micrograms per milliliter in the appropriate medium. Treat the cells for 15 minutes in a cell culture incubator at 37 degrees Celsius.
Prior to micro-irradiation, use a micropipette to rinse the cells twice with medium without phenol red, to ensure maximal exposure of the cells to the 405 nanometer laser. Any confocal microscope equipped with the 405 nanometer laser can be used for this method. Users need to carefully calibrate their system to achieve physiological levels of DNA damage.
We describe how to do this in the detailed protocol. Select a suitable laser scanning confocal microscope equipped with a 405 nanometer laser. Turn on the microscope and the environmental chamber.
Select fields with well-distributed cells, adjust the focus, and register their position to facilitate image acquisition after the immunofluorescence protocol. If using a fluorescently-labeled protein for live imaging, acquire at least one image that will serve as a pre-damage reference point, to monitor the kinetics of protein recruitment after micro-irradiation. For immunofluorescence experiments using BBET-labeled cells adjust the focus on the cells using the 405 nanometer laser at the lowest laser power sufficient to visualize the cells, to avoid unwarranted DNA damage.
If using cells expressing the fluorescently-labeled protein of interest, select a focal plane with representative fluorescence intensity. Configure the microscope for the micro-irradiation step using the fluorescence-recovery after photobleaching module of the system. Using the FRAP module of the microscope system, select regions of interest that will be micro-irradiated.
Once this is done, micro-irradiate the cells. Typically, chromatin-associated factors will be recruited to double-stranded breaks within two minutes. Resection of the breaks by nucleases will generate single-stranded DNA, and promote the recruitment of homologous recombination factors which become detectable 10 minutes after micro-irradiation.
After micro-irradiation, place the multi-well slides back into the incubator at 37 degrees Celsius with 5%CO2 for the required duration prior to processing samples for immunofluorescence. Or, proceed immediately with live time-lapse imaging as detailed in the text protocol. To begin analysis, open the acquired images in Fiji.
Select the Damage Analyzer from the pull-down plug-ins menu in the micro-irradiation analysis suite. Define the time units and the time interval between frames, then register the image series using the StackReg algorithm. Follow the prompts to define a background region of interest, or ROI, in each micro-irradiated cell.
Also, define non-irradiated and irradiated ROIs in each micro-irradiated cell. Once all ROI data have been collected over the full time-lapse series, save the file created by the plug-in containing the results as a comma-delimited file. This file contains measurements of the mean intensity for each time point and each ROI.
Perform the analysis using the plug-in for all micro-irradiated cells, and graph the relative enrichment at micro-irradiated regions over time. This step should be repeated for at least 15 to 30 cells, and the mean enrichment and the standard errors of the mean for each data point should be calculated and plotted. Using a micropipette, carefully remove the media from the wells of the multi-well microscopy slides.
Immediately wash the cells with 500 microliters of IF washing solution by changing the solution twice without waiting. Then, incubate on ice for five minutes, with 500 microliters of ice-cold IF pre-and post-extraction solution. Swirl gently by hand for 15 to 30 seconds to perform the pre-extraction step that removes most cytoplasmic and nucleoplasmic proteins, and maximizes the signal from chromatin and DNA associated factors.
Next, wash the cells twice with 500 microliters of IF washing solution. Then, add 500 microliters of IF fixation solution, and incubate for 15 minutes at room temperature. After washing twice with 500 microliters of IF washing solution, incubate on ice for five minutes in 500 microliters of ice-cold IF pre-and post-extraction solution.
Swirl the slide gently by hand to perform the permeabilization step. Wash the cells twice with 500 microliters of IF washing solution. Then, incubate at least 30 minutes at room temperature with 500 microliters of IF blocking solution.
Remove the blocking solution using a micropipette and add 250 microliters of primary antibody solution. Then, incubate the cells overnight in a humidified chamber at 4 degrees Celsius. The next day, wash the cells four times for five minutes in 500 microliters of IF washing solution with gentle agitation.
Then, incubate the cells for one hour at 37 degrees Celsius with 250 microliters of secondary antibodies diluted in blocking solution in a humidified chamber. Protect the samples from light upon addition of fluorescently-labeled secondary antibodies. Then, wash the cells four times each for five minutes in a 500 microliters of IF washing solution with gentle agitation.
Incubate the cells at room temperature for five minutes in 500 microliters of 1X PBS, containing one microgram per milliliter of DAPI to stain the nuclei. After washing the cells twice with 500 microliters of 1X PBS, leave cells in 500 microliters of 1X PBS solution for imaging. Using gridded slides or registered stage positions, find the micro-irradiated cells using a well-characterized DNA damage marker.
Image the multi-well culture slides in all relevant channels using the appropriate settings on a laser scanning confocal microscope. Laser micro-irradiation of pre-sensitized cells leads to the production of DNA double-stranded breaks, which are resected by nucleases to generate single-stranded DNA. Proteins or modifications that spread to large chromatin domains appear as broad stripes within two minutes of micro-irradiation.
This is exemplified by the pattern observed with antibodies targeting the phosphorylated histone variant H2A. X.The single-stranded DNA can be visualized at later time points as punctate foci using antibodies against replication protein A.Using this method, it is evident that 53BP1 is recruited onto large chromatin domains, whereas the PRP19 E3 ubiquitin ligase is recruited onto RPA-coded single-stranded DNA. Transfection of plasmids encoding proteins tagged with fluorescent reporters, followed by micro-irradiation, allows the monitoring of protein recruitment to DNA lesions in real time.
Micro-irradiation of BRDU pre-sensitized cells transfected with plasmids expressing EGFP, or RPA32 fused to GFP, show that RPA32-GFP but not EGFP alone, is rapidly recruited to the single-stranded DNA created by resection of the double-stranded DNA breaks. This graph shows the quantification of the RPA32 GFP signal enrichment over time at micro-irradiated loci from 15 independent cells analyzed with the micro-irradiation analysis Fiji plug-in. Once mastered, this technique can be done in approximately four hours for live imaging of 10 to 15 individual cells expressing fluorescently-labeled proteins, and within two days when immunofluorescence is used to monitor protein recruitment at micro-irradiated loci.
After watching this video, you should have a good understanding of how to configure a laser scanning microscope and use it to micro-irradiate cells and monitor the recruitment of DNA repair factors to sites of damage, both in real time and in fixed cells. While attempting this procedure, it's important to remember that the conditions described here generate double-stranded breaks, but also other types of lesions. But the user can alter micro-irradiation and pre-sensitization methods to create specific types of lesions relevant to their research.
Following this procedure, depletion of established DNA damage response factors are mutation of specific domains in the protein of interest can be performed to provide additional insight into the recruitment mechanism of these factors at DNA breaks. Don't forget that working with lasers and paraformaldehyde can be extremely hazardous, and precautions such as wearing personal protection equipment, and working in a chemical hood when required should always be taken while performing this procedure.
