12/21/2023 0 Comments Bliss font kitHere, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.Įukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription (3) quantification of DSBs through unique molecular identifiers and (4) easy scalability and multiplexing. Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. 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. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. 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. We developed RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks.
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