Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

As the study uses human cells, the work was approved by the Ethics Committee of the Institute of Biomedical Science at the University of São Paulo (ICB-USP, approval number #48347515.3.00005467) for the research with human samples.

NOTE: The protocols described here were used in previous publications with minor modifications^14,16,28. Here the focus is on the use of ultraviolet light C (UVC) as a DNA damaging agent. However, other DNA damaging agents such as cisplatin and hydroxyurea have also been successfully employed²⁸. This protocol can be performed in an array of cell lines, including U2OS, HEK293T, human fibroblasts, and others^14,28,29. It is essential to determine the experimental question before performing this protocol. The DNA fiber assay with the S1 nuclease (called S1 Fiber hereafter) is used to detect the presence of post-replicative ssDNA gaps, while the gap-filling (or PRR) assay is performed to quantify gap-filling events in the G2 phase. Thus, if analyzing the presence of post-replicative ssDNA gaps, the investigator should perform section 2 (S1 Fiber experimental setup) and proceed directly to section 4 (DNA fiber preparation by spreading) of the protocol. If analyzing gap-filling events, the investigator should proceed directly to section 3 (Gap-filling (PRR) experimental setup).

1. Reagents and setup

1. Thymidine analogs: Resuspend 5-Iodo-2'-deoxyuridine (IdU) and 5-Chloro-2'-deoxyuridine (CldU) in 1 N NH₄OH to a final concentration of 100 mM. Filter the analogs through a sterile syringe filter (0.2 µm), aliquot, and store them at -20 C.
2. Antibodies: Primaries – anti-BrdU rat (Biorad) and anti-BrdU mouse (BD); Secondaries – anti-rat Alexa Fluor 488 and anti-mouse Alexa Fluor 594.
3. Cellular permeabilization CSK100 buffer for the S1 Fiber: Add 100 mM NaCl, 10 mM MOPS [pH 7], 3 mM MgCl₂ [pH 7.2], 300 mM sucrose and 0.5% Triton X-100 in distilled H₂O.
4. S1 nuclease buffer [pH 4.6]: Add 30 mM Sodium Acetate [pH 4.6], 10 mM Zinc Acetate, 50 mM NaCl and 5% Glycerol in H₂O.
   NOTE: The S1 nuclease buffer must have a final pH of 4.6, as the S1 activity drops 50% at pH > 4.9.
5. Aliquot and store at -20 °C the S1 nuclease purified from A. oryzae pre-diluted in S1 nuclease dilution buffer provided by the manufacturer.
6. Reconstitute the nocodazole in DMSO to a final concentration of 2 mM for gap-filling. assay.
7. Lysis buffer: Add 200 mM Tris-HCl [pH 7.5], 50 mM EDTA and 0.5 % SDS in H₂O.
8. Prepare phosphate-buffered saline (PBS).
9. Prepare PBS-T by adding 0.1% Tween-20 in PBS.
10. Prepare 5% Bovine Serum Albumin (BSA) in PBS.
11. Prepare 0.1% BSA in PBS.
12. Prepare PBS-T-BSA by adding 1% BSA in PBS-T.
    NOTE: Use an epifluorescent microscope with a mercury or xenon lamp with a 510 nm wavelength and 42 nm bandwidth GFP (FITC, Alexa Fluor 488) emission filter and a 624 nm wavelength and 40 nm bandwidth Texas Red (Alexa Fluor 594) emission filter or a confocal microscope with a 488-nm line (blue) laser to detect green fluorescence (detected between 510 and 560 nm) and a 561-nm line (yellow) laser to detect red fluorescence (detected between 575 and 680 nm). Immersion objective (40x, 63x or 100x).

2. S1 Fiber experimental setup (2 days)

1. Day 1: Plate 4 wells for each cell line: ± UVC and ± S1 nuclease with cells at 70-90% confluency.
   ​NOTE: The experiment can be done in a format as small as a 12-well plate. In this case, all solutions are used at 0.5 mL/well.
2. Day 2: Prepare fresh IdU at 20 µM and CldU at 200 µM in pre-warmed (37 °C) cell culture media.
3. Aspirate the culture media and immediately add the media with 20 µM IdU and incubate the cells at 37 °C, 5% CO₂ (cell incubator) for precisely 20 min.
4. Quickly wash the cells with pre-warmed (37 °C) PBS twice.
5. Irradiate the cells with 20 J/m² UVC. Use untreated cells as controls.
6. Immediately add the media with 200 µM CldU and incubate the cells at 37 °C, 5% CO₂ (cell incubator) for precisely 60 min.
   NOTE: IdU and CldU labeling can be interchanged, but the concentration of the second analog should be 5-10 times higher than the first analog. Timing of the analog incubation can be adapted to the genotoxic agent used, but the time of the second analog incubation should be long enough to ensure enough time for gap formation^14,16. If using a DNA damaging drug, add the drug together with CldU^28,29,30.
7. Wash the cells with pre-warmed (37 °C) PBS twice.
8. Permeabilize the cells with the CSK100 buffer for 8-10 min at room temperature (RT).
   NOTE: Successful permeabilization can be checked under a brightfield microscope where only nuclei should be observable
9. Carefully wash the nuclei with PBS (pour 0.5 mL down the side of each well, just for a few seconds).
10. Carefully wash the nuclei with S1 buffer (pour 0.5 mL down the side of each well, just for a few seconds).
11. Incubate the nuclei with S1 buffer with S1 nuclease (20 U/mL) or without (as a control) for 30 min at 37 °C (cell incubator).
12. Aspirate the S1 buffer and add 0.1% BSA in PBS.
13. Scrape the nuclei and transfer them to an appropriately annotated 1.5 mL tube. Keep tubes on ice all the time.
    NOTE: Successful detachment of nuclei can be checked under a brightfield microscope.
14. Pellet the nuclei: centrifuge at ~4600 x g for 5 min, at 4 °C.
    ​NOTE: Different from the standard DNA fiber assay, the nuclei cannot be counted, so consider the initial number of cells plated. Alternatively, an extra well that was not subjected to permeabilization can be used to estimate the cell count using a hemocytometer or a cell counter.
15. Resuspend the pellets well in PBS at a concentration of ~1500 cells/µL, place the tubes on ice and proceed to DNA fiber preparation by spreading (section 4) protocol immediately

3. Gap-filling (PRR) experimental setup (3 days)

1. Day 1: Plate 2 wells for each cell line: ± UVC with cells at ~70 % confluency.
2. Day 2: Prepare fresh IdU at 20 µM in pre-warmed (37 °C) cell culture media.
3. Irradiate the cells with 20 J/m² UVC.
4. Immediately add the media with 20 µM IdU and incubate the cells at 37 °C, 5% CO2 (cell incubator) for precisely 60 min.
5. Wash the cells with pre-warmed (37 °C) PBS twice.
6. Immediately add the media with 200 ng/mL nocodazole and incubate the cells at 37 °C, 5% CO₂ (cell incubator) for 12-24 h (timing of this step must be kept consistent between experiments).
   ​NOTE: The concentration of nocodazole might need to be adapted according to the cell line used. If using a genotoxic drug, incubate the cells with IdU ± drug²⁸.
7. Day 3: Aspirate the culture media, add the media with 20 µM CldU + nocodazole, and incubate for the last 4 h of nocodazole treatment, at 37 °C, 5% CO₂ (cell incubator).
8. Wash the cells with pre-warmed (37 °C) PBS.
9. Add trypsin and incubate for 1-2 min at 37 °C, 5% CO₂ (cell incubator) to detach cells.
10. Add the same volume of cell culture media and collect cells (media + trypsin) in an appropriately annotated 1.5 mL tube. Keep the tubes on ice all the time.
11. Count the cells using a hemocytometer or a cell counter.
12. Pellet the cells: centrifuge at ~350 x g for 5 min, at 4 °C.
13. Remove the supernatant and resuspend the cells in PBS at ~1500 cells/µL. Keep the cells on ice and start DNA fiber preparation by spreading protocol immediately (Day 3 or 4).

4. DNA fiber preparation by spreading (~1 h for 12 slides)

1. Annotate the microscope slides with a carbon pencil and place them flat on a tray.
2. Tap the bottom of the tube to ensure homogenization.
3. Add 2 µL of cells on the top of the corresponding slide.
   NOTE: Two drops can be added, one on the top of the slide and another in the middle of the slide.
4. Add 6 µL of the lysis buffer and lyse the cells by pipetting up and down about 5 times (avoid making bubbles).
5. Pull the drop a little bit towards the bottom of the slide with the pipette tip (to guide the path of the drop spreading).
6. Incubate for 5 min at RT to lyse the nuclei.
   NOTE: The volume of lysis buffer and the time of lysis can be adjusted if the lysate dries too quickly or, alternatively, does not dry enough. Lysis buffer volume can vary from 5-7 µL and timing can fall between 4-10 min.
7. Tilt the slide at around 20-40° and allow the drop to spread to the bottom of the slide at a constant, low speed.
8. Allow the slide to dry at RT in the dark (10-15 min).
9. Freshly prepare the fixing solution: Mix methanol: glacial acetic acid at a 3:1 ratio.
10. Immerse the slides in a jar containing the fixing solution and incubate for 5 min at RT to fix DNA onto the slides.
    ​CAUTION: Methanol is highly toxic and should be kept under a chemical hood. Discard in an appropriate container.
11. Dry the slides at RT in the dark and store at 4 °C protected from light until staining.

5. Immunostaining of DNA fibers (~6 h)

1. Wash the slides with PBS twice for 5 min.
2. Denature the DNA with freshly prepared 2.5 M HCl for 40-60 min at RT.
   CAUTION: To prepare 2.5 M HCl, pour acid in water and never the opposite. This is an exothermic reaction so it will warm up slightly.
3. Wash the slides with PBS three times for 5 min.
4. Block with 5% BSA (can be stored at 20 °C for up to three uses) previously warmed at 37 °C for 30-60 min at 37 °C.
5. Remove the excess liquid from slides by gently tapping on a paper.
6. Add 30 µL of primary antibodies (mouse anti-BrdU 1/20 (for IdU) and rat anti-BrdU 1/100 (for CldU) diluted in PBS-T-BSA) to the slides dropwise and place a coverslip on top. Incubate for 1-1.5 h at RT in a dark, humid chamber.
7. Place the slides in a jar with PBS for 1-2 min, then carefully remove the coverslips.
8. Wash with PBS-T in a jar three times for 5 min, then place the slides in PBS.
9. Remove the excess liquid by gently tapping on a paper.
10. Add 30 µL of secondary antibodies (anti-mouse Alexa Fluor 594 and anti-rat Alexa Fluor 488 diluted at 1/75 in PBS-T-BSA.) to the slides dropwise and place a coverslip on top. Incubate for 45-60 min at RT in a dark, humid chamber.
11. Place the slides in a jar with PBS for 1-2 min, then carefully remove the coverslips.
12. Wash with PBS-T in a jar three times for 5 min, then place the slides in PBS.
13. Remove the excess liquid by gently tapping on a paper.
14. Add 20 µL of the mounting reagent dropwise to the slides and place a coverslip on top. Avoid bubbles.
15. Let the slides dry at RT in the dark, then store at 4 °C (or -20 °C for longer conservation).

6. Image acquisition and analysis (~1 h per sample)

NOTE: An epifluorescence microscope can be used for both protocols. However, for the gap-filling (PRR) assay, using a confocal microscope is highly recommended for the increased resolution of the images.

1. Place a drop of immersion oil (microscope specific) near the top of the slide where the cells were lysed and find the focus using the green channel by searching green dots in the background (40x, 63x, and 100x objectives can be used).
2. Find the main concentration of fibers and move to the edges to find well-spread non-overlapping individual fibers using only one channel to select the regions for the pictures and avoid potential bias.
3. Take around 15-20 pictures per slide alongside the entire slide at the green and red channels and merge the two channels to obtain bicolor DNA fibers.
   NOTE: If using a confocal microscope, take pictures at high resolution (1024 x 1024 pixels) and use pinhole values close to 1 for both red and green fluorescence.
4. Use ImageJ (free software provided by the NIH, https://imagej.nih.gov/ij/) for IdU and CldU tract length measurements.
   1. Open a picture by dragging it into the ImageJ box.
   2. Use the scale bar generated by the microscope software (appropriately calibrated) to convert the length to micrometers. Use the Straight-line tool by selecting the 5^th icon from left to right on the ImageJ box to measure the scale bar in the picture provided micrometers and obtain a measurement in pixels. Click on Analyze > Set Scale and replace the "distance in pixels" with the appropriate number and the "unit of length" to µm.
   3. On the ImageJ box, select the Segmented Line tool by left-clicking on the 5^th icon from left to right and then selecting the second option from top to bottom.
   4. Draw the exact path of the red (IdU) or green (CldU) tract by left-clicking and stop the drawing by right-clicking. Push the M button on the keyboard to measure the length.
   5. For the S1 Fiber, only analyze red-green (bicolor fibers)¹⁶. Measure red and green tract lengths from at least 150 fibers from different pictures by keeping track of the red and green tracts measurements for each individual fiber.
   6. For the gap-filling assay, only measure the length of IdU (red) tracts that contain at least 1 CldU (green) patch but no continuous CldU staining. Score at least 10-15 IdU tracts from different pictures with at least 1 CldU patch.
      NOTE: Other software can be used for the analyses, such as Zeiss LSM Image Browser.
5. For the S1 Fibers, plot the IdU tracts length, CldU tracts length, and ratios (CldU/IdU or vice-versa) in separate graphics as scatter dot plots with medians.
6. Measure gap-filling events as units of "per-kilobase density", divide the total number of CldU (PRR, green) patches on a given length of IdU (red) tract by the total length of the red tract in kilobases (considering 1 µm = 2.59 kilobases³¹). Then plot the data as scatter dot plots with medians.
7. In both experiments, perform statistical analysis between two samples using Mann-Whitney (nonparametric test) and between more than two samples using Kruskal-Wallis followed by Dunn's multiple comparisons test.