Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

This methodology video introduces the crucial steps of immunostaining and chromatin immunoprecipitation protocols regularly used to study DNA damage-related cellular process and to visualize and quantify the recruitment of proteins implicated in DNA repair. This technique provides powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus, as well as their post-translational modifications necessary for the fine-tuned regulation during DNA repair. Since these techniques can be applied for studying different cellular processes, they have potential relevance if a laser microirradiation or the nucleus-based DNA damaging inducing systems are used.

Recently, several powerful tools have been developed to induce site-specific DNA damage that can be used to extend our understanding of DNA repair. Applying biochemical and genetic approaches, these utensils are essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. Moreover, these techniques allow for the study of these processes at single cell resolution using both fixed and living cells.

Maintain U2OS cells in monolayers in DMEM culture medium supplemented with 10%fetal calf serum, 2 millimolar glutamine, and the 1%antibiotic-antimycotic solution. Grow cells in a humidified 5%carbon dioxide environment at 37 degree until 80%confluence is achieved, renewing medium every two to three days. Aspirate the medium and wash the cells with PBS.

Detach cells with Trypsin-EDTA solution. When the cells detach, stop the activity of the trypsine by adding culture media to the cells, yielding a cell suspension. Count the cells using a cell counting chamber.

Plate 20, 000 cells per milliliter per well on the 24-well plate with sterile 12 millimeter round cover slips in each well. Incubate cells for 24 hours at 37 degree in a humidified 5%carbon dioxide environment to allow attachment onto the cover slips. Treat the cells with 10 nanogram per milliliter neocarzinostatin or use the appropriate method to induce double-strand breaks via endonucleuse-based systems.

Incubate cells for one to eight hours at 37 degree in a humidified 5%carbon dioxide environment to follow the kinetics of the DNA repair. Fix cells with 4%formaldehyde PBS solution for 20 minutes at 25 degree. Remove the fixing solution and wash cells three times with PBS for five minutes each.

Remove the PBS and add 0.2%Triton X dissolved in PBS. Incubate the samples for 20 minutes. Wash the cells three times with PBS.

Block non-specific binding with 5%BSA diluted in BSA-PBST and incubate the samples for at least 20 minutes. Add the proper amount of primary antibody diluted in 1%BSA-PBST solution. Place each cover slip upside down onto a parafilm or 10 microliter droplet of the diluted antibody.

Incubate the samples in a humidity chamber for one and the half hour at four degree. Place the cover slips back being side up into the 24-well plate and the wash three times with PBS. Add the proper amount of secondary antibody diluted in 1%BSA-PBST.

Place each cover slip upside down onto parafilm over a 10 microliter droplet of the diluted antibody. Incubate the samples in a humidity chamber at four degree for an hour. Place the cover slips back being set up into the 24-well plate and wash three times with PBS.

Before removal of the last PBS washing solution, gently take out the cover slips with the half of the tweezer and needle, and then put them upside down onto the glass slides with droplets of mounting medium. Chromatin immunoprecipitation is a widely used technique to identify binding patterns of proteins to DNA. To reveal the occupancy of a desire repair protein around the DSB, a specific antibody is used to immunoprecipitate the protein of interest from the chromatin preparation together with the DNA region to which it is bound.

Furthermore, ChIP has been commonly applied to map the presence of specific post-translational modification in use by DSBs at a given genomic locus. Approximately 20 million cells per milliliter should be cultured in a 15 centimeter dish for each sample. Remove culture medium and wash the cells twice with ice cold PBS.

Fix the cells with 1%formaldehyde-PBS solution. Place the plate on an orbital shaker and agitate gently for 20 minutes. Stop fixation with glycine to reach a final concentration of 125 millimolar and incubate on an orbiter shaker with gentle agitation for five minutes at 25 degree.

Place the plate on ice and wash twice with ice cold PBS. Scrape the cells in ice cold PBS and transfer them into 15 milliliter conical tubes. Centrifuge the cells at 5, 000 rpm for five minutes at four degree.

Carefully aspirate the supernatant and resuspend the pellet in two milliliter cell lysis buffer and incubate on ice for 10 minutes. Centrifuge at 5, 000 rpm for five minutes at four degree. Carefully discard the supernatant and resuspend the pellet in 500 to 1, 500 microliter nuclear lysis buffer and incubate on ice for 30 to 60 minutes.

Transfer the lysate into a polystyrene conical tube suitable for sonication. Sonicate the lysate to shear the DNA to an average fragment size of 300 to 1000 base pairs. Prepare Dynabeads for the pre-cleaning and immunoprecipitation steps.

Wash beads twice for 10 minutes at four degree with RIPA buffer. Resuspend Dynabeads in the same volume of RIPA buffer as you took out from the original stock solution in steps 3.1. Pre-clear 25 to 30 microgram chromatin of each sample with four microliter Dynabeads via rotation for one to two hours at four degree.

Precipitate the Dynabeads with a magnet and transfer the supernatant to a new Eppendorf tube. Add the appropriate amount of antibody to each chromatin sample and rotate overnight at four degree. Next day, add 40 microliter of washed Dynabeads to each sample and incubate them overnight rotating at four degree.

Wash once with 300 microliter low salt buffer for 10 minutes with rotation at four degree. Wash once with 300 microliter high salt buffer for 10 minutes with rotation at four degree. Wash once with 300 microliter lithium chloride buffer for 10 minutes with rotation at four degree.

Wash twice with 300 microliter TE for 10 minutes with rotation for the first wash at four degree and the second wash at 25 degree. Add 200 microliter elution buffer to the beads and incubate them at 65 degree in a thermo-shaker for 15 minutes with continuous shaking. Transfer the supernatant to a new tube and elute beads again in 200 microliter elution buffer.

Add 500 microgram per milliliter proteinase-K and half percent SDS and incubate the samples at 50 degree for two hours. Perform chloroform extraction and transfer the upper aqueous phase to a new Eppendorf tube. Add two and a half volumes of absolute ethanol and 0.1 volume three molar sodium acetate pH 5.2.

Incubate for at least 20 minutes at minus 80 degree. Remove the ethanol and air dry the pellet. Resuspend the pellet in 50 microliter TE.To study their special distribution on DNA, chromatin immunoprecipitation should be applied.

A typical experimental results obtained by ChIP-qPCR is presented in this figure, which demonstrates the temporal enrichment of gamma histone AX as a response to I-PpoI-induced induced DNA damage. On the left part of the image, the timely detected gamma histone AX signal is shown at the break side, while on the right part, gamma histone AX distribution is presented at the control gene region at which demonstrated breaks have not been induced. Although DNA repair is a relatively recent research field, our knowledge is rapidly increasing with the help of both biochemical and microscopic methods.

Nonetheless, biochemical techniques, such as a Western blot, immunoprecipitation, and mass spectrometry require a large amount of cells. The study repair processes represent a snapshot of the desired cell population. The microscopic field has been revolutionized by high-resolution techniques, such as super resolution microscope, which allows the visualization of DNA damage-induced cellular processes at the nucleosomal level, as well as ensuring the accurate mapping of protein co-localization.

On the other hand, chromatin immunoprecipitation is a powerful tool, especially by combining it with sequencing methods to visualize its genome-wide binding pattern of a desired DNA repair related proteins and double strand breaks in either a chromatin or heterochromatin regions.