Which Comes First, The Deformation or the Binding?

Abstract

Which comes first, the deformation or the binding?High specificity of protein-DNA interaction is often related with specific deformation of the binding site. B-Z transition is the most dramatic structural change induced by protein-DNA interaction, where some segment of DNA abruptly changes from the right-handed B-DNA to the left-handed Z-DNA by the help of specific proteins.Here, we report single-molecule FRET studies on protein-induced Z-DNA formation. DNA duplexes with six CG-repeats were prepared. To monitor the conformational dynamics of the CG-repeat, we labeled Cy3 and Cy5 at each end of the CG-repeat. Surface-immobilized DNA molecules did not show any structural dynamics in normal physiological conditions. When a Z-DNA inducing protein, Zα, was added to the buffer solution, however, fluorescence intensity increased abruptly without any accompanying FRET change. Abrupt FRET change occurred with time delay (∼10 minute at 25°C on average). When the proteins were washed out, molecules didn’t recover the original FRET value for more than 3 hours, but molecules without the FRET change readily recovered their original fluorescence intensity. From these result, we conclude that Zα protein weakly interact with B-DNA, but the interaction becomes extremely strong once Z-DNA is formed.Next, we prepared a DNA duplex with methylated cytosine in the CG repeat. With millimolar Ni2+ in the buffer solution, we observed the intrinsic B-Z transition dynamics, and Z-DNA stabilization by Zα proteins. The transition time from B-DNA to Z-DNA, however, was not affected by the presence of Zα proteins, which strongly support that Zα protein induces Z-DNA by passively trapping Z-DNA structure transiently formed by the intrinsic B-Z transition dynamics. Even though we cannot directly observe Z-DNA, Z-DNA's are actually waiting there inside the cell to play their biological roles on time.