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

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.Author summary: Damage to the genetic code embedded in an organism’s DNA can result in mutation or cell death which, in turn, can lead to disease and dysfunction. DNA damage is the main cause of many human diseases including cancer, and some forms of neurodegeneration and immune dysfunction. DNA double strand breaks (DSBs), which occur a handful of times in each replicating cell each day, are especially deleterious due to the difficulty of their repair. The main endogenous cause of DSBs is the breakdown of DNA replication forks however there is increasing evidence that damage and stress at these forks can also result in unbroken intermediate structures which avoid DSB formation such as fork regression. We have developed and applied new assays for labelling and visualizing damaged replication forks using super-resolution microscopy. This has enabled us to differentiate between broken and unbroken forks and to discern the different proteins that are recruited for DSB and regressed fork repair. Our data further demonstrate that these assays are widely applicable to DNA damage research and offer a new approach to mapping the spatiotemporal repair of individual damage events inside cells.