Super-resolution visualization of distinct stalled and broken replication fork structures

• Published in: PLoS genetics Vol. 16; no. 12; p. e1009256
• Main Authors: Whelan, Donna R, Lee, Wei Ting C, Marks, Frances, Kong, Yu Tina, Yin, Yandong, Rothenberg, Eli
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.12.2020; Public Library of Science (PLoS)
• Subjects: Biology and life sciences; Cell cycle; Cell Line, Tumor; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA biosynthesis; DNA Breaks, Double-Stranded; DNA damage; DNA Replication; Electromagnetic radiation; Fluorescence microscopy; Humans; Microscopy; MRE11 Homologue Protein - metabolism; Physical sciences; Physiological aspects; Proteins; Rad51 Recombinase - metabolism; Rad52 DNA Repair and Recombination Protein - metabolism; RecQ Helicases - metabolism; Recruitment; Replication; Replication fork; Research and Analysis Methods; Single Molecule Imaging - methods; Spatial discrimination; Transcription; Visualization; United Kingdom
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1009256

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.