DNA knots, catenanes, and replication intermediates via high-resolution AFM

<p dir="ltr">This repository contains the raw and processed atomic force microscopy image data for the journal paper titled; "Under or Over? Tracing Complex DNA Structures with High Resolution Atomic Force Microscopy" (bioRxiv) or "Quantifting complexity in DNA Structures with High Resolution Atomic Force Microscopy" Nature Communications, which uses a new automated computational methods to trace complex knotted, catenated, and theta-curve DNA topologies provided by the <i>E.coli</i> Xer recombination system, and stalled replication intermediates from <i>Xenopus</i> egg extracts.</p><p dir="ltr">The raw and processed image files can be found for each dataset (knots and catenanes, and replication intermediates) within the "AFM data" folder.</p><p dir="ltr">The outputs from the TopoStats software can be found in the "AFM analysis" folders.</p><p dir="ltr">The analysis scripts used to plot graphs from outputs of the TopoStats software are found in "Analysis scripts.</p><p dir="ltr">The topology of DNA plays a crucial role in the regulation of cellular processes and genome stability. Despite its significance, DNA topology remains challenging to determine due to the length and conformational complexity of individual topologically constrained DNA molecules. We demonstrate unparalleled resolution of complex DNA topologies using Atomic Force Microscopy (AFM) in aqueous conditions. We present a new high-throughput automated pipeline to determine DNA topology from raw AFM images, using deep-learning methods to trace the backbone of individual DNA molecules and identify crossing points. Our pipeline efficiently determines which segment passes over which, including the handling of challenging crossings, where the path of each molecule may be harder to resolve. We demonstrate the wider applicability of our tracing method by determining the structure of stalled replication intermediates from Xenopus egg extracts, including theta structures and late replication products. By developing new methodologies to accurately trace the DNA path through every crossing, we determine the topology of plasmids, knots and catenanes from the E. coli Xer recombination system. In doing so we uncover a recurrent depositional effect and reveal its origins using coarse-grained simulations. Our approach is broadly applicable to a range of nucleic acid structures, including those which interact with proteins, and opens avenues for understanding fundamental biological processes which are regulated by or affect DNA topology.</p>