Under or Over? Tracing Complex DNA Structures with High Resolution Atomic Force Microscopy

Abstract

ABSTRACTThe topology of DNA plays a crucial role in the regulation of cellular processes and genome stability. Despite its significance, DNA topology is challenging to explicitly determine due to the length and conformational complexity of individual topologically constrained DNA molecules. We demonstrate unparalleled resolution of complex DNA topologies in aqueous solutions, achieving resolution of the double helix around two intertwined molecules using atomic force microscopy (AFM). 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 strands and identify crossing points. Our pipeline efficiently handles complications with tracing which arise at these crossings, where the path of each molecule is harder to resolve, to determine which molecule passes over which. By accurately tracing the DNA path through every crossing, we determine the topology of plasmids, knots and catenanes from theE. coliXer recombination system. In doing so we uncover a recurrent depositional effect and reveal its origins using coarse-grained simulations. Finally, we demonstrate the wide applicability of this method by determining the structure of stalled replication intermediates fromXenopusegg extracts, including theta structures and late replication products. Our approach can be applied to a range of DNA and RNA structures, including those interacting with proteins, and opens avenues for understanding fundamental biological processes which are regulated by or affect DNA topology.