Examining the Impact of Supercoiling on Sequence-Dependent DNA Structure and Flexibility

Abstract

Supercoiling has been shown to promote DNA structural transitions from canonical B-form DNA to alternative DNA structures; one of the most dramatic being to the left-handed Z-form DNA. Supercoiling pays essential roles in cellular biology; it is required to package genomic DNA into confined regions within cells, and negative supercoiling is thought to promote DNA strand separation during DNA replication and transcription initiation. Moreover, promotion of the B-DNA to Z-DNA transition by negative supercoiling occurs under physiological conditions, suggesting the B-to-Z transition is one way to alleviate negative superhelical stress. Namely, sequences that favor Z-DNA often exist upstream of transcriptional start sites and their specific location has been shown to regular transcription levels in vivo. Our objective is to examine the impact of supercoiling on Z-DNA formation and base pair opening in DNA at nucleotide and atomic resolution. A major challenge in studying supercoiled DNA at the atomic scale is that supercoiled closed-circular DNA plasmids contain thousands of nucleotides and therefore push the limitations for characterizations by biophysical techniques such as NMR and X-Ray crystallography. Recently, DNA minicircles of less than 100 base pairs have been synthesized to contain superhelical stress as a model system in biochemical studies to investigate the influence of superhelical forces on many aspects of DNA such as energetic stability and protein binding. We propose to overcome experimental limitations in atomic studies of supercoiled DNA through the development of a novel biochemical assay, as well as the application of recent advances in NMR spectroscopy. Using these new tools, we will test the hypothesis that supercoiling allows access to non-B-form structures, including Z-DNA and looped out bases, in a sequence-dependent manner.