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https://www.selleckchem.com/B-Raf.html 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.Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used it to trigger site-specific replication fork stalling and homologous recombination (HR) at a defined chromosomal locus in mammalian cells. By comparing HR responses induced at the Tus-Ter RFB with those induced by a site-specific double-strand break (DSB), we have begun to uncover how the mechanisms of mammalian stalled fork repair differ from those underlying the repair of a replication-independent DSB. Here, we outline how to transiently express the Tus protein in mES cells, how to use flow cytometry to score conservative and aberrant repair outcomes, and how to quantify distinct repair outcomes in response to replication fork stalling at the inducible Tus-Ter chromosomal RFB.Repair of double-strand DNA

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