Tackling twist with TopoStats

Session

AFM in Life Sciences

Authors

Dr Alice Pyne (1)

Affiliations

1. University of Sheffield

Keywords

Atomic force microscopy, DNA, open science, python, software

Abstract text

The complexity of cellular DNA is a consequence of its innate flexibility, compaction in the nucleus, and manipulation of its structure by DNA-processing enzymes, resulting in a vast conformational landscape. Within this landscape DNA adopts intricate structures, conformations and topologies and is frequently maintained under torsional or superhelical stress, in an under- or over- wound state. There is a growing appreciation that the topological complexity of DNA in the genome affects DNA-protein interactions key to cell viability. Beyond modulating interactions with protein machinery, DNA underwinding influences the formation of alternative DNA structures including G-quadruplexes (G4s). The effect of torsional stress and topological complexity on DNA are hard to quantify, as they cause changes to DNA structure in the nanometre size range, which cannot be observed in linear DNA and are ‘invisible’ to techniques that rely on averaging. New technological developments for high resolution atomic force microscopy (AFM) allow us to visualise single biomolecules in liquid with sub-molecular resolution. This allows us to quantify the twist, writhe, and topology of single DNA molecules in liquid as they ‘explore’ their complex conformational space. 

A rate-limiting step in the widespread adoption of AFM to solve problems inaccessible to the traditional tools of structural biology is a lack of open software pipelines to analyse the increasing volumes of data produced. Automated analysis tools/software pipelines for AFM would reduce reliance on an experienced researcher, minimise selection bias and facilitate the growth of AFM as a quantitative imaging technique. We have developed TopoStats (www.github.com/AFM-SPM/TopoStats), an open-source Python utility that loads raw AFM data and handles data cleaning and processing through to identification and characterisation of individual biomolecules [1]. 

We use TopoStats to quantify the effect of supercoiling on DNA structure, demonstrating that DNA under superhelical stress is far richer in structure, e.g., containing kinks and defects, than can be observed in short linear sequences [2]. We have built on this foundation to develop tools that can accurately and automatically pinpoint crossings in complex DNA structures such as knots and catenanes. These new image analysis routines can almost unambiguously automatically identify under- and over-passing segments of DNA at each DNA crossing, thus allowing full identification of the topology of each DNA molecule. Finally, we expand our toolkit beyond single DNA molecules, to show that the DNA-binding protein NDP52 binds specifically and with high affinity to double-stranded DNA and that this interaction leads to changes in DNA structure. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation [3].

References

[1]             Beton, JG et al. Methods 193, 68-79 (2021) 

[2]             Pyne, ALB*, Noy A* et al. Nature Communications. 12, 1053 (2021)

[3]             Dos Santos, A et al. BioRxiv (2022)