This study investigates the role of ATP-dependent chromatin remodeling proteins in regulating gene expression by altering DNA conformation. Using circular dichroism (CD) spectroscopy, the interactions between chromatin remodelers and DNA are analyzed.
ATP-dependent chromatin remodeling proteins regulate gene expression in tightly-packed chromatin by altering the DNA conformation in an ATP-dependent manner.
To study chromatin remodeler-DNA interactions using circular dichroism, or CD, spectroscopy, take a solution of double-stranded DNA oligonucleotides. Heat to denature the oligonucleotides, and place on ice.
Fast cooling causes the DNA to renature while forming secondary structures. The double-to-single-strand transition region provides an optimal binding site for the chromatin remodelers.
Add the heat-cooled DNA into a cuvette. Add a buffer containing ATP-dependent chromatin remodeler, ATP, and magnesium ions.
The remodeler binds to the DNA, followed by the binding of magnesium ion-complexed ATP to the protein — activating it. The activated remodeler hydrolyzes ATP — inducing a stem-loop conformation in the DNA.
Pass left and right circularly-polarized light — with a 90° phase difference — through the sample. Optically chiral DNA absorbs the left and right circularly-polarized light to different extents.
The differential absorption, termed circular dichroism, results in the transmitted light being elliptically polarized, which is measured as ellipticity. Two positive peaks at specific wavelengths in the CD spectra confirm the stem-loop structure.
Add EDTA to chelate the magnesium ions, inhibiting the ATPase activity.
A change in the CD spectra upon chelation — showing a negative peak and a broad positive peak at specific wavelengths — indicates the disruption of the stem-loop conformation in the absence of ATP hydrolysis.
Collect the CD spectra in high-transparency quartz cuvettes. Use either rectangular or cylindrical cuvettes. To clean the cuvettes, wash them with water several times, then, take a scan of the water or buffer in the cuvette to check whether it is clean.
For the reactions, use PAGE-purified DNA oligonucleotides. For fast-cooling, heat the DNA at 94 degrees Celsius for 3 minutes on the heating block and immediately cool it on ice. For a slow-cooling, after heating the DNA at 94 degrees Celsius for 3 minutes, allow it to cool to room temperature at a rate of 1 degree Celsius per minute.
To record the baseline spectra, set up a total of 5 control reactions, one by one, in 1.5-milliliter centrifuge tubes. Keep the reaction volume at 300 microliters in all the reactions.
To record the CD spectra, set up a total of five experimental reactions one by one in 1.5-milliliter centrifuge tubes.
To record the scan, turn on the gas, and switch on the CD spectrometer. After 10 to 15 minutes, switch on the lamp, switch on the water bath, and set the holder temperature at 37 degrees Celsius.
Next, open the CD spectrum software and set the temperature to 37 degrees Celsius, the wavelength range at 180 to 300 nanometers, the time per point to 0.5 seconds, and the scan number to 5. Then, click on the Pro-Data Viewer, make a new file, and rename it with the details of the experiment and the date.
Next, mix the baseline and experimental reactions by pipetting, and carefully transfer the reaction mixes one by one to the cuvette, ensuring that there are no air bubbles.
If performing a time-course experiment, incubate the reactions at 37 degrees Celsius for the required time. Then, take the scan. Add EDTA to the buffer containing the DNA, ATP, magnesium, and protein to stop ATP hydrolysis. To completely inhibit the ATPase activity, increase the EDTA concentration and incubation time.
Subtract the baselines from the corresponding reactions in the software, and smoothen the data either in the CD spectrum software or in the data-plotting software. Then, plot a graph of wavelength against mean residue ellipticity and analyze the peaks.
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