Investigation of the Formation of Z-DNA and Other Non-Canonical DNA Conformations Using a Combined Spectroscopic and Biochemical Approach

Abstract

Genomic sequences of DNA not only code for proteins, but also unique structural conformations encompassing a wide variety of helical and base-pairing geometries. Formation of these sequence-specific conformations is driven by a complex network of cellular interactions including ions, proteins, and supercoiling forces that constantly bombard the DNA double helix. One particular conformation Z-DNA, composed of a left-handed helix, is believed to play important roles in gene expression and regulation. Formation of Z-DNA within the genome results in the inversion of right-handed B-DNA and creation of distorted junctions at the intersection of B and Z-form helices. However, much is still unknown about the sequence-dependence of the B-to-Z transition and B/Z junction formation. Here we use a wide range of spectroscopic and biochemical methods to characterize the formation of Z-DNA, B/Z junctions, and other non-canonical DNA conformations. NMR relaxation dispersion allows for an unprecedented insight into lowly populated conformations. First, we carry out simulations to explore the limits of which systems can be quantitatively characterized by R1rho relaxation dispersion. Using relaxation dispersion experiments, we show that sites near or at B/Z junctions have a high intrinsic propensity in B-DNA to form non-canonical conformations. By combining NMR dynamics measurements with CD measurements of the B-to-Z transition, we show that mutations that diminish local flexibility at B/Z junctions also reduce the propensity to undergo the B-to-Z transition. To better characterize the role of B/Z junction formation in the B-to-Z transition, we develop a combined CD and fluorescence spectroscopic approach for quantitatively assessing the formation of B/Z junctions within mixtures of B and Z-DNA. Our studies show that the thermodynamics of B/Z junction formation can significantly influence the B-to-Z transition, allowing for the incorporation of unfavorable sequences into Z-DNA in order to achieve the most favorable B/Z junction. These new surprising preferences for Z-DNA formation may expand the sequence-space predicted to be available to Z-DNA in genomes. Finally, we have initiated studies on supercoiled DNA using small minicircles and carry out experiments to probe how sequence-specific mutations, shown to have a dramatic effect on relaxed linear DNA, influence the properties of supercoiled DNA.