Our protocol opens new possibilities to investigate how replication occurs on damaged DNA. The techniques can detect the formation and repair of single-stranded DNA gaps, opposite the DNA lesion. The S1 nuclease cleaves the single-stranded DNA and allows the detection of post-replicative gaps.
This approach was first used by Dr.Meneghini and Dr.Cordiero-Stone in the 1970s in Brazil, and recently adapted by us to the DNA fiber assay. Using the technique described here to study the formation and repair of post-replicative gaps can provide insights into the mechanism of DNA repair and replication stress response that maintain genome integrity. For both protocols described here, the S1 fiber and the gap-filling assays, It is crucial to include all the controls to ensure accurate interpretation of the data.
The procedure will be demonstrated by Davi J.Martins, a PhD student from our lab, and Dr.Annabel Quinet, a collaborator who previously developed these techniques when working in our group. To begin with, aspirate the culture media and immediately add the culture media with 20-micromolar of IDU media to the cells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for precisely 20 minutes.
Then, quickly wash the cells with pre-warmed PBS twice and irradiate them with 20 joules per meter square UV-C. Use untreated cells as controls. Immediately add the media with 200-micromolar CldU to the cells and incubate them at 37 degrees Celsius and 5%carbon dioxide for precisely 60 minutes.
After that, wash the cells with pre-warmed PBS twice and permeabilize them with the CSK 100 buffer for two to 10 minutes according to the cell line at room temperature. After CSK permeabilization, the nuclei with almost no cytoplasm can be seen compared to those before the permeabilization. Carefully pour PBS down the side of each well to wash the nuclei with PBS.
Then, wash the nuclei with S1 buffer. Incubate the nuclei in the S1 buffer with 20 units per milliliter S1 nuclease, and without the S1 nuclease, for 30 minutes at 37 degrees Celsius. Next, aspirate the S1 buffer and add 0.1%BSA in PBS.
Scrape the nuclei and transfer them to an approximately annotated 1.5-milliliter tube. Keep the tubes on ice all the time. Centrifuge the nuclei at around 4, 600 times G for five minutes at four degrees Celsius.
Resuspend the pellets in PBS at a concentration of approximately 1, 500 cells per microliter. Place the tubes on ice and immediately proceed to DNA fiber preparation by spreading protocol. Annotate the microscope slides with a carbon pencil and place them flat on a tray.
Tap the bottom of the tube to ensure homogenization and add two microliters of cells on the top of the corresponding slide. Add six microliters of the lysis buffer and lyse the cells by pipetting up and down about five times. Use the pipette tip to pull the drop slightly towards the bottom of the slide and incubate for five minutes at room temperature to lyse the nuclei.
Tilt the slide at around 20 to 40 degrees and allow the drop to spread to the bottom of the slide at a constant low speed. Allow the slide to dry at room temperature in the dark for 10 to 15 minutes. Then, immerse them in a jar containing the freshly prepared fixing solution and incubate for five minutes at room temperature to fix DNA onto the slides.
Wash the slides with PBS twice for five minutes and denature the DNA with freshly prepared 2.5-molars cloridric acid for 40 to 60 minutes at room temperature. Wash the slides with PBS three times for five minutes and block with 5%BSA previously warmed for 30 to 60 minutes at 37 degrees Celsius. Gently tap on the paper to remove the excess liquid from the slides.
Add 30 microliters of primary antibodies to the slides dropwise and place a cover slip on top. Incubate for 1 to 1.5 hours at room temperature in a dark, humid chamber. Place the slides in a jar with PBS for one to two minutes and then carefully remove the cover slips.
Wash with PBST in a jar three times for five minutes and place the slides in PBS. Remove the excess liquid by gently tapping on a paper and add 30 microliters of the secondary antibodies to the slides dropwise. Place a cover slip on top.
Incubate for 45 to 60 minutes at room temperature in a dark and humid chamber. Repeat the steps for washing the slides and removing the excess PBS once more and add 20 microliters of the mounting reagent dropwise to the slides. Place a cover slip on top and avoid bubbles.
Place a drop of immersion oil near the top of the slide where the cells were lysed. Use the green channel and search the green dots in the background to find the focus. Open the Leica Application Suite software, click on Acquire, and select the green channel.
Adjust the settings of the green channel before analysis. Then, change to the red channel and adjust the settings. Find the main concentration of fibers and move to the edges to find the well spread, non-overlapping, individual fibers.
Use only one channel to select the regions for the pictures and avoid potential bias. Click on the Acquire Overlay icon and adjust the software for the correct microscope magnification. Take around 15 to 20 pictures per slide alongside the entire slide at the green channel and then at the red channel.
The two channels are now merged and the created overlay is moved to a unique folder. It is possible to see the fibers with red and green colors. Open the ImageJ software, left click on the fifth icon, select the second option, and then select the Segmented Line tool.
Left click to draw the exact path of the red or green tract and right click to stop the drawing. Push the M button on the keyboard to measure the length. For the S1 fiber, keep track of the red and green tracts measurements for each fiber and measure red and green tract lengths from at least 150 fibers from different pictures.
An S1 fiber is shown in these representative images. Control-nascent DNA tract without the gaps and impervious to cleavage by the S1 nuclease is shown here. DNA tract positive for gaps cleaved by the S1 nuclease resulted in a shorter CldU tract length.
A differential labeling scheme extensively described in the text manuscript can be performed to label gap-filling events. In this assay, named gap-filling assay, longer IDU tracks with at least one CldU patch or fewer CldU patches correspond with low gap-filling density. Shorter IDU tracts, with at least one CldU patch, and longer IDU tracts with more CldU patches, correspond with high gap-filling density.
The permeabilization step of this protocol is crucial for the S1 nuclease to act on the gaps formed during the replication of damaged DNA. The S1 nuclease can also be used in the comet assay protocol to detect the gaps formed during the replication of damaged DNA that persists after treatment. The S1 fiber assay paved the way for the new paradigm that post-replicative gaps can cause cancer vulnerability, particularly in breast and ovarian cancers carrying mutations in BRCA1 and 2 genes.
