DNA damage response is indispensable for life as DNA is constantly exposed to various damaging agents. Lack of the repair leads to genomes'instability and diseases. We developed a method based on biological property of the RPA2 to visualize single strand DNA tract during DNA repair in different cell cycle stages.
Traditional method for single strand DNA detection utilize BrdU antibodies. This approach can be used for actively replicating cells. Additionally, this antibody tend to cross-react with other nucleotide analogs limiting their use.
Unlike BrdU, our approach has a higher signal to noise ratio, making it usable in every cycle phase. Understanding the action of different DNA repair pathways in various cell cycle phases and potentially non-replicating cells is crucial. Our protocol provides a new approach and tool to visualize single-stranded DNA foci, where classical approaches utilizing BrdU are limited.
A comprehensive understanding of DNA repair processes could serve as therapeutic targets to treat cancer, aging and neurological diseases. Most of our cells are terminally differentiated and therefore do not replicate and divide. It is crucial to gain a better understanding of how these cells repair various DNA lesions.
Therefore, our laboratory will be mainly focusing on post replicative DNA repair processes and developing new tools to study them. To begin sterilize tweezers with 70%ethanol. Place a single glass cover slip into a well of a 24-well plate.
Add 500 microliters of Vitronectin solution into each well containing the cover slips. After incubation, remove the coating solution and wash the cover slips with one milliliter of PBS. Incubate the RPE1 cells in one milliliter of 0.05%Trypsin at 37 degrees Celsius.
To detach the serum starved cells from a tissue cultured treated plate, resuspend the RPE1 cells in six milliliters of DMEM medium to inactivate the Trypsin. Next, centrifuge the cells at 150 G at room temperature for five minutes. Then resuspend the cells in one milliliter of culturing medium.
Seed 500 microliters of the cell suspension onto the coated cover slip. Pulse the cells with 10 micromoles of EdU for 30 minutes at 37 degrees Celsius. Remove the medium containing EdU and chase the cells with 10 micromoles of thymidine for 10 minutes at 37 degrees Celsius.
After removing the thymidine medium, treat the cells with 250 micromoles of hydrogen peroxide for one hour. To perform pre-extraction, incubate the washed cells in one milliliter of CSK extraction buffer for five minutes at room temperature. Then fix the cells in 0.5 milliliters of 3.6%paraformaldehyde solution for 10 minutes at room temperature.
Next, wash the cells in one milliliter of PBS containing 0.05%Triton X-100 to remove the paraformaldehyde. After permeabilization is complete, remove the solution and wash the cells two times with one milliliter of blocking buffer. Then add one milliliter of blocking buffer and gently rock the plate for 10 minutes at room temperature.
Add 500 microliters of Click reaction cocktail containing Picolyl Azide-647 after removing the blocking buffer. Then wash the cells two times with PBS containing 0.05%Triton X-100 for 10 minutes at room temperature. Now incubate the washed cells in one milliliter of blocking buffer for 30 minutes at room temperature.
Then incubate the cells in primary antibody for two hours at room temperature in 250 to 500 microliters of blocking buffer. Wash the cells two times with Triton X PBS. Then wash the cells in blocking buffer.
Now apply the secondary antibody in 250 to 500 microliters of blocking buffer at room temperature for two hours with gentle rocking. Once incubation is complete, wash the cells in PBS and blocking buffer. Counter stain nuclei of the washed cells in DAPI for 10 minutes at room temperature.
Then wash cells with PBS for five minutes at room temperature. Dip the cover slips into distilled water to remove salt crystals. Then mount the cover glass onto microscope slides in 10 microliters of mounting medium.
Store the stained slides at four degrees Celsius. To capture images, use an epifluorescent microscope with routine filter sets for DAPI, FITC, and Cy5 channels. Open the image files in the Fiji software.
To make the nuclear masks using the DAPI staining, first, open the DAPI image. Select Process followed by Enhance Contrast and set the saturated pixel to 0.35. Next, press Process again and choose Binary, followed by Convert to Mask.
Click on Process and Binary again. Then press Fill Holes. Click Analyze Particles under Analyze and set the size to 10-Infinity.
Next, click on Show All in the ROI Manager To find the RPA2 foci, first, open the RPA2 image. Click on Process and choose Find Maxima. Then set the prominence to a value that highlights the foci.
Click the Measure button in ROI Manager. Then divide the value in the RawIntDen column by 255 to calculate the total number of nuclear single-stranded DNA foci. The addition of serum containing media to starved cells for six hours resulted in reentry into the cell cycle.
Cell synchronization was confirmed with the EdU incorporation assay and the lack of DNA replication in G1 phase. Hydrogen peroxide and neocarzinostatin treatment resulted in BrdU foci detection only in the S phase cells. Neither the untreated nor the treated samples showed colocalization of EdU and BrdU, demonstrating no antibody cross reactivity in our approach.
RPA2 staining showed neocarzinostatin and hydrogen peroxide dependent foci formation, not only in the S phase, but also in other cell cycle phases. The RPA2 antibody also detected naturally occurring single-stranded DNA formation during replication in the absence of exogenous genotoxic stress. A significant increase in the RPA2 foci was observed in the cells in G1 phase upon hydrogen peroxide treatment.
