This technique facilitates the visualization of replisome antigen-tagged DNA adduct encounters and can be extended to the interactions of any protein with a DNA structure or lesion amenable to immunodetection. This method can be used to assess interaction frequencies within individual cells, rather than across a population, and allows the visualization of cell cycle-dependent interactions in situ without damaging cells. Demonstrating the procedure will be Ishani Majumdar, a post-doc from the Seidman Laboratory.
On the day before the experiment, seed 1.5 times 10 to the fifth cells in two milliliters of medium onto 35-millimeter glass bottom culture dishes pre-treated with cell adhesive solution. The next day, replace the supernatant in each dish with 37 degrees Celsius Five micromolar Dig TMP-supplemented medium to the 50 to 70%confluent cell cultures and return the plates to the cell culture incubator for 30 minutes to allow the Dig TMP to equilibrate. While the cells are incubating, prewarm a UV box to 37 degrees Celsius.
At the end of the equilibration, transfer the plates to the pre-warmed box to expose the cells to a five-minute three-joule-per-square-centimeter UVA light dose and replace the supernatants with two milliliter of fresh pre-warmed culture medium before returning the plates to the cell culture incubator for one hour. At the end of the incubation, gently wash the cultures with PBS and fix the cells with one milliliter of 0.1%formaldehyde in PBS for five minutes at room temperature. At the end of the incubation, wash the cells one time with PBS before treating the cells two times with 80 microliters of cytoskeleton extraction buffer supplemented with RNase for five minutes at room temperature per treatment to remove the cytoplasm.
After the second incubation, wash the cells with two milliliter of PBS three times before fixing the cells with one milliliter of 4%formaldehyde in PBS for 10 minutes at room temperature. After the second fixation, wash the cells three times with PBS as demonstrated, followed by treatment with one milliliter of cold 100%methanol for 20 minutes at minus 20 degrees Celsius. At the end of the incubation, wash the cells three times in two milliliters of PBS, followed by a 10 minute incubation in 80 microliters of five millimolar Triton X-100 at four degrees Celsius.
At the end of the incubation, treat the cells with 100 microliters of five millimolar EDTA in PBS, supplemented with one microliter of 100-milligram-per-milliliter of RNase A for 30 minutes at 37 degrees Celsius. At the end of the incubation, wash the cells three times in PBS before storing them in 5%bovine serum albumin and 10%donkey serum in PBS in a humid chamber overnight at four degrees Celsius. To perform a proximity ligation assay, add 40 microliters of the primary antibody solution of interest to the center of each plate and incubate the plates in the humid chamber for one hour at 37 degrees Celsius.
At the end of the incubation, wash the cells three times with two milliliters of PBST before adding 40 microliters of freshly prepared PLA probe solution to each dish for a one hour incubation in the cell culture incubator. At the end of the incubation, wash the cells with three 10-minute washes in two milliliters of buffer A on a tilting platform at room temperature. After the last wash, add 40 microliters of freshly prepared ligation mix to each plate for a 30-minute incubation at 37 degrees Celsius, followed by three two-minute washes in buffer A on the tilting platform.
After the last wash, add 40 microliters of freshly prepared amplification solution to each plate for a 100-minute incubation at 37 degrees Celsius in a humid chamber. At the end of the incubation, wash the cells six times with fresh buffer B for 10 minutes per wash on the tilting platform. After the last buffer B wash, wash the cells one time with 0.01x buffer B for one minute at room temperature before labeling with the appropriate secondary antibodies in the humid chamber for 30 minutes at 37 degrees Celsius.
At the end of the incubation, wash the samples with three 10-minute washes on a tilting platform in PBST at room temperature before mounting them in mounting medium supplemented with DAPI. within four days of mounting, image at least 100 observations per sample or condition cells on an epifluorescence or confocal microscope, using the same exposure settings for both experimental and control samples. The Dig tag can be visualized by immunofluorescence to reveal the presence of interstrand crosslinks throughout the nucleus.
To visualize the interaction of replisomes with interstrand crosslinks, PLA can be applied to visualize the frequency of association of MCM5 and the Dig tag, one hour after interstrand crosslink introduction. As replication stress activates ATR kinase, the PLA between pMCM two serine 108 and the Dig tag is also positive. The PLA results from individual nuclei can be presented in a three-dimensional reconstruction allowing the visualization of replisome interstrand crosslink encounters throughout the nucleus.
Replication Fork analysis indicates that interstrand crosslink encounters demonstrate an unexpected replication restart phenomenon, as the proteins of the GINS complex fail to exhibit PLA signal with the interstrand crosslinks. In contrast, the assay with CD 45 is positive, indicating that the other locking protein is retained. When cells are incubated with an ATR inhibitor, the restart is completely suppressed and the GINS Dig PLAs strongly positive.
CSK+R treatment is essential for reducing background signal. Be sure to coat the plates with a cell adhesive and to prefix with 0.1%FA or the cells will peel off the plate.
