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Chromosome Choreography: Chromosome translocations arise through the illegitimate pairing of broken chromosome ends and are commonly found in many cancers. Roukos et al. (p. 660 ) used ultrahigh-throughput time-lapse imaging on human tissue culture cells containing marked chromosomes to capture very rare translocation events. Double-strand breaks in the DNA underwent an initial “partner search,” with a fraction of the ends moving into spatial proximity to each other, which resulted in persistent pairing and the merging of DNA repair foci. Most paired ends arose from breaks in close proximity, but occasionally translocations formed from more distantly positioned breaks. Proteins of the DNA repair machinery could influence the pairing and/or translocation process.

This Protocol from the Misteli lab describes how to determine the cell cycle stage of each individual cell in a population by using fluorescence microscopy and automated image analysis software. Progression through the cell cycle is one of the most fundamental features of cells. Studies of the cell cycle have traditionally relied on the analysis of populations, and they often require specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unperturbed cells. We describe a protocol, suitable for use in high-resolution imaging approaches, for determining cell cycle staging of individual cells by measuring their DNA content by fluorescence microscopy. The approach is based on the accurate quantification by image analysis of the integrated nuclear intensity of cells stained with a DNA dye, and it can be used in combination with several histochemical methods. We describe and provide the algorithms for two automated image analysis pipelines and the derivation of cell cycle profiles with both commercial and open-source software. This 1–2-d protocol is applicable to adherent cells, and it is adaptable for use with several DNA dyes.

Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. We developed RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.