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Genome-wide detection of DNA double-strand breaks by in-suspension BLISS

ABSTRACT: Genome-wide detection of DNA double-strand breaks by in-suspension BLISS

PROVIDER: PRJNA607701 | ENA |

REPOSITORIES: ENA

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Genome-wide detection of DNA double-strand breaks by in-suspension BLISS

Project description:sBLISS (in-suspension Break Labeling In Situ and Sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs), in any cell type that can be brought into suspension. After in situ labeling, DSB ends are linearly amplified followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, followed by a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which circumvents the need to work with delicate coverslips as in the original BLISS, (ii) the possibility for adaptation to a high-throughput robotics or single-cell workflow, and (iii) its flexibility and applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks, and it is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.

2020-09-30 | GSE145598 | GEO

Nucleosomes protect DNA from irradiation-induced double strand breaks in living cells [BLISS human]

Project description:The number and position of breaks using BLISS-seq was measured in MCF-7 cells upon irradiation with 10 Gy and with or without the use of the antioxidant (N-acetylcysteine, NAC, 10mM).

2020-07-07 | GSE150016 | GEO

Nucleosomes protect DNA from irradiation-induced double strand breaks in living cells (BLISS)

Project description:The depletion of HMGB1 reduces the deposition of histones on naked DNA (Celona et al, 2011). in primary MEFs and yeast. We verified that the downmodulation of HMGB1 decreases histone level also in a model of murine mesothelioma. HMGB1 was KD in Murine Mesothelioma AB22 cells by the expression of a shRNA. Nucleosome positioning and occupancy were tested by ATAC-seq. Since a reduced amount of nucleosomes increase the cell sensitivity to gamma irradiatio, we tested the number and position of breaks using BLISS-seq.

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Nucleosomes protect DNA from irradiation-induced double strand breaks in living cells (BLISS)

Project description:Nucleosomes protect DNA from irradiation-induced double strand breaks in living cells (BLISS)

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Nucleosomes protect DNA from irradiation-induced double strand breaks in living cells [BLISS human]

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Double-strand breaks in spo11 mutants

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Doxorubicin induces double-strand breaks at CpG island promoters

Project description:Doxorubicin is a widely used chemotherapeutic drug that intercalates between DNA base-pairs and posions Topoisomerase II, although the mechanistic basis for cell killing remains speculative. Here we show that both anthracyclines and Topoisomerase II poison cause enhanced DNA double-strand breaks around CpG island promoters of active genes genome-wide. We propose that torsion-based enhancement of nucleosome turnover exposes promoter DNA, ultimately causing DNA breaks around promoters that contributes to cell killing. We have analyzed mouse squamous cell carcinoma cells treated with doxorubicin, aclarubicin and etoposide. The direct in situ Breaks Labeling, Enrichment on Streptavidin (BLESS, PMID 23503052) method was used for mapping DNA double-strand breaks genome-wide.

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Genome-wide maps of double-strand breaks

Project description:Mapping physiological double strand breaks (DSBs) in cancer cells uncovers transcription-coupled repair mechanism at oncogenic super-enhancers in which RAD51 of the homologous recombination pathway plays a key role supporting the hyper-transcription of related oncogenes.

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Genome-wide maps of double-strand breaks

Project description:Genome-wide maps of double-strand breaks

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Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts [BLISS]

Project description:RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localisation to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localisation is poorly understood. Here we show that the topoisomerase-II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-induced nucleolar antisense RNAs (diNARs) in human cancer cells. diNARs originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in ab RRM1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signaling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

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