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

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 E.coli Xer recombination system, and stalled replication intermediates from Xenopus egg extracts.
The raw and processed image files can be found for each dataset (knots and catenanes, and replication intermediates) within the "AFM data" folder.
The outputs from the TopoStats software can be found in the "AFM analysis" folders.
The analysis scripts used to plot graphs from outputs of the TopoStats software are found in "Analysis scripts.
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.

本仓库包含对应两篇期刊论文的原始与处理后的原子力显微镜（Atomic Force Microscopy, AFM）图像数据，两篇论文分别为发表于bioRxiv的《Under or Over？利用高分辨率原子力显微镜示踪复杂DNA结构》，以及发表于Nature Communications的《高分辨率原子力显微镜下量化DNA结构的复杂性》。该研究采用全新的自动化计算方法，对大肠杆菌（E. coli）Xer重组系统产生的复杂打结、连环及θ环DNA拓扑结构，以及非洲爪蟾卵提取物中的停滞复制中间体进行示踪。
原始与处理后的图像文件可在"AFM data"文件夹下的各数据集（打结结构、连环结构与复制中间体）中获取。TopoStats软件的输出结果存放在"AFM analysis"文件夹中。用于基于TopoStats软件输出结果绘制图表的分析脚本，可在"Analysis scripts"文件夹内找到。
DNA拓扑结构在细胞过程调控与基因组稳定性维持中发挥关键作用。尽管其重要性不言而喻，但由于受拓扑约束的DNA分子长度较长且构象复杂，DNA拓扑结构的解析仍颇具挑战。本研究证明，在水相条件下利用原子力显微镜（AFM）可实现复杂DNA拓扑结构的超高分辨率解析。我们开发了一套全新的高通量自动化分析流程，可从原始AFM图像中解析DNA拓扑结构：通过深度学习方法追踪单个DNA分子的骨架并识别交叉点，可高效判定各DNA链的上下交叉关系，甚至可处理难以分辨的复杂交叉场景。我们通过解析非洲爪蟾卵提取物中的停滞复制中间体（包括θ结构与晚期复制产物），验证了该示踪方法的广泛适用性。此外，我们还开发了可精准追踪每一处交叉点处DNA路径的新方法，据此解析了大肠杆菌（E. coli）Xer重组系统中的质粒、打结结构与连环结构的拓扑属性。在此过程中，我们发现了一种重复性的沉积效应，并通过粗粒度模拟（coarse-grained simulations）揭示了其成因。本研究方法可广泛应用于各类核酸结构（包括与蛋白质相互作用的核酸结构），为理解受DNA拓扑结构调控或影响DNA拓扑结构的基础生物学过程提供了新的研究方向。