Single-molecule super-resolution microscopy (SRM) combines single-molecule detection with spatial resolutions tenfold improved over conventional confocal microscopy. These two key advantages make it possible to visualize individual DNA replication and damage events within the cellular context of fixed cells. This in turn engenders the ability to decipher variations between individual replicative and damage species within a single nucleus, elucidating different subpopulations of stress and repair events. Here, we describe the protocol for combining SRM with novel labeling and damage assays to characterize DNA double-strand break (DSB) induction at stressed replication forks (RFs) and subsequent repair by homologous recombination (HR). These assays enable spatiotemporal mapping of DNA damage response and repair proteins to establish their in vivo function and interactions, as well as detailed characterization of specific dysfunctions in HR caused by drugs or mutations of interest.
Keywords: DNA damage response; DNA double-strand break; DNA replication; Homologous recombination; Super resolution.