Keynote Talk  - Friday, 17 September I 14:00 PM (CEST)

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Dr. Alice Pyne: "Base-pair resolution analysis of the effect of supercoiling on DNA structure and interactions"

University of Sheffield, Sir Robert Hadfield Building, Sheffield, S1 3JD, UK

Email: a.l.pyne@sheffield.ac.uk

Understanding how DNA behaves in its cellular environment is a challenge of complexity, which can be enhanced by a better understanding of the fundamental properties of DNA. In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. We overcome these limitations by combining high-resolution AFM¹ and atomistic MD simulations to resolve the structure, conformation and dynamics of supercoiled DNA to the base-pair level (Figure 1)².

We use DNA minicircles, only twice the persistence length of DNA, to probe the structure and function of negatively-supercoiled DNA. These minicircles are small enough to be  simulated at the atomistic level by MD and to be visualized at high (double-helix) resolution by AFM experiments in solution 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 flexibility³. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. 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.

Figure 1.  Structural diversity in supercoiled DNA minicircles. Synergistic high-resolution AFM images (a-d) and MD snapshots (e) of natively supercoiled DNA minicircles show striking structural diversity in natively supercoiled DNA minicircles. Scale bars: 10 nm. Height scale (inset): 2.5 nm for all images

 

References:

1.           Pyne, A., Thompson, R., Leung, C., Roy, D. & Hoogenboom, B. W. Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic Force Microscopy. Small 10, 3257–3261 (2014).

2.           Pyne, A. L. B. et al. Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides. Nature Communications 12, 1053 (2021).

3.           Beton, J. G. et al. TopoStats – A program for automated tracing of biomolecules from AFM images. Methods (2021) doi:10.1016/j.ymeth.2021.01.008.

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