S1 nuclease fiber assay

S1 Nuclease DNA Fiber Assays

The following protocol is established to directly visualize replication fork dynamics and replication associated gaps at a single molecular DNA fiber level.

Stage 1:Treatment with the nucleotide analogs and S1 nuclease.

1. Seed 1.5X 10⁶ cells in a 10cm plate with regular culture media.

2. Next day, remove the media and treat the cells with complete media containing 50uM 5-Iodo-2'-deoxyuridine (IdU) for 30mins. The plate should be kept in the incubator (37°C) covered with a foil to protect it from light. 

3. After 30mins, remove the media and wash well with 1X PBS twice.

4. Now again treat the cells with complete media containing 50uM 5-Chloro-2'-deoxyuridine (CldU) and 0.5mM Hydroxyurea (HU) for 1hr [Here one can use any form of replication stress other than HU if required, upon proper dose and time optimization]. Remember to keep the plate in the incubator (37°C) covered with a foil to protect it from light.

5. Repeat step 3.

6. Subsequently, cells are permeabilized with CSK buffer (100 mM NaCl, 10 mM MOPS, 3 mM MgCl2 pH 7.2, 300 mM sucrose, 0.5% Triton X-100) at room temperature (RT) for 8mins. Remember to use enough volume of the CSK buffer to cover the plate. Do not exceed this step for more than 8mins.

7. Carefully wash the plate with 1X PBS once.

8. Now repeat the washing once with S1 buffer (30 mM Sodium Acetate pH 4.6, 10 mM Zinc Acetate, 5% Glycerol, 50 mM NaCl).

9. Once the cells are primed with the S1 buffer, treat them with S1 nuclease (20U/ml) made in S1 buffer for 30mins at 37°C. Do not exceed 30mins. [For the control plate, treat the cells with only S1 buffer without S1 nuclease for the same duration of time].

10. Remove the S1 buffer and add 5ml of 1X PBS +0.1%BSA in 10cm plate and scrape the cells. Do not trypsinize. Collect the cells in a pre-chilled 15ml tube, put on ice and centrifuge at 7000 rpm for 5mins at 4°C to collect the cell pellet. 

11. Remove the supernatant and resuspend the cell pellet again in 1X PBS + 0.1%BSA such that the final cell suspension is 1500 cells/ul. Make sure to resuspend the pellet well and perform these steps on ice and immediately proceed to preparing the DNA fiber slides.

Stage 2:Preparing the DNA fiber slides (Remember to perform these steps in dark and avoid light)

12. Mix the samples well by pipetting up and down.

13. Add 2.5ul of cell suspension on one end of a positively charged slide.

14. Add 7.5ul of lysis buffer (200mM Tris-HCl pH 7.5, 50mM EDTA, 0.5% SDS) to the same spot where you added the cells suspension and gently pipette up and down 4-5 times to mix the two and lyse the cells. Incubate the drop for exactly 7-8mins at RT and not more.

15. Tilt the slide at 45° angle to allow the drop that now contains the DNA from the cells to run down the slide.

16. Wait and let the drop to air dry for 15-20mins which will result in a white smudge or line across the slide.

17. Fix the DNA by keeping the slide in a coplin jar with freshly prepared and ice-cold Methanol:Acetic Acid (3:1) for 5mins.

18. Let the slides dry and store at 4°C or proceed with the staining.

Stage 3:Staining the DNA fibers (Remember to perform these steps in dark and avoid light)

18. Wash the slides twice with 1X PBS for 5mins each in a coplin jar.

19. Denature the DNA for 1hr by treating with 2.5M HCl at RT in a coplin jar.

20. Wash the slides three times with 1X PBS for 5mins each to neutralize the acid in a coplin jar.

21. Treat the slides with blocking buffer (3%BSA made in 1X PBS + 0.1% Triton-X) for 30-45mins at RT in a coplin jar.

22. Wash twice with 1X PBS and proceed with primary antibody staining in a coplin jar.

23. Incubate with 50ul of primary antibodies for 2.5hrs at RT in a humid chamber. Remember to make the antibody dilutions in the blocking buffer. Make sure that the antibody covers the entire surface of the slide. This can be achieved by gently placing a parafilm cut in the size of the slide over as a coverslip to avoid drying. 

Antibodies: IdU [Becton Dickinson 347580 – Mouse monoclonal anti BrdU 1:100]

                 CldU [Abcam ab6328 – Rat anti BrdU 1:100]

24. Put 500ul of 1X PBS on the slides to gently remove the parafilm.

25. Wash the slides with 1X PBS for 5mins for a total of three times.

26. Incubate with 50ul of secondary antibodies for 1hr at RT in a humid chamber. Follow step 23 to avoid drying.

Antibodies: IdU [Alexa 488 – Goat anti mouse 1:200]

                  CldU [Alexa 594 – Goat anti rat 1:200]

27. Repeat steps 24 and 25.

28. Dry the slides and add 20ul of Prolong gold Antifade (Invitrogen, P36930) and add a coverslip.

29. Let the slide dry at RT in the dark and then store at 4°C (at -20°C for longer storage).

30. Finally, the visualization of green and/or red signals by fluorescence microscopy (Axioplan 2 imaging, Zeiss).

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