The Circular Dichroism Spectroscopy Technique to Study DNA-Protein Interactions

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