Year: 2020
Type: article
Abstract: 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.
Source: PLoS Genetics
Authors: Donna R. Whelan, Wei Ting C. Lee, Frances Marks, Yu Tina Kong, Yandong Yin, Eli Rothenberg
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
Awards: G1935969957, Mechanism and Fidelity of RAG mediated DNA recombination, DNA Replication Stress: Characterizing ground zero for genomic instability, G9000275336, Mechanisms of Human DNA Double-Strand Break Repair via Quantitative Single-Molecule Imaging, Polymerase theta, genome instability, and cancer
OpenAlex ID: W3115169046