It plays a fundamental role in the formation and stability of looped DNA structures 8 and DNA R-loops 9, and influences the placement of RNA guide sequences by the CRISPR-Cas9 gene editing toolkit 10. In eukaryotes, supercoiling generated by transcription is implicated in the regulation of oncogenes such as c-Myc 7. Supercoiling operates synergistically with nuclear-associated proteins to regulate bacterial gene expression 6. In prokaryotes, genomic DNA has an average density of supercoiling, σ (∆Lk/original Lk) of ~−0.06 5. The conformational response to this stress is called negative supercoiling, partitioned between untwisting of the helix (change in twist Tw) and a coiling deformation of the DNA backbone (writhe Wr) 1, 2, 3, 4.
Negative superhelical stress results from a reduction in the number of links (Lk) between the two strands of a closed-circular DNA (a negative ∆Lk). Genomic DNA is often subjected to torsional stress, which can both over- and under-wind the DNA double helix 1, 2, 3. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures.
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