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An atlas of endogenous DNA double-strand breaks arising during human neural cell fate determination.

ABSTRACT: Endogenous DNA double-strand breaks (DSBs) occurring in neural cells have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs). Currently, a genomic map of endogenous DSBs arising during human neurogenesis is missing. Here, we applied in-suspension Breaks Labeling In Situ and Sequencing (sBLISS), RNA-Seq, and Hi-C to chart the genomic landscape of DSBs and relate it to gene expression and genome architecture in 2D cultures of human neuroepithelial stem cells (NES), neural progenitor cells (NPC), and post-mitotic neural cells (NEU). Endogenous DSBs were enriched at the promoter and along the gene body of transcriptionally active genes, at the borders of topologically associating domains (TADs), and around chromatin loop anchors. NDD risk genes harbored significantly more DSBs in comparison to other protein-coding genes, especially in NEU cells. We provide sBLISS, RNA-Seq, and Hi-C datasets for each differentiation stage, and all the scripts needed to reproduce our analyses. Our datasets and tools represent a unique resource that can be harnessed to investigate the role of genome fragility in the pathogenesis of NDDs.

SUBMITTER: Ballarino R

PROVIDER: S-EPMC9276747 | biostudies-literature |

REPOSITORIES: biostudies-literature

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An atlas of endogenous DNA double-strand breaks arising during neural cell fate determination

Project description:Profiling of endogenous DNA double-strand breaks during neural cell fate determination using sBLISS.

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Replication-independent endogenous DNA double-strand breaks in Saccharomyces cerevisiae model.

Project description:Without exposure to any DNA-damaging agents, non-dividing eukaryotic cells carry endogenous DNA double-strand breaks (EDSBs), or Replication-Independent (RIND)-EDSBs. In human cells, RIND-EDSBs are enriched in the methylated heterochromatic areas of the genome and are repaired by an ATM-dependent non-homologous end-joining pathway (NHEJ). Here, we showed that Saccharomyces cerevisiae similarly possess RIND-EDSBs. Various levels of EDSBs were detected during different phases of the cell cycle, including G0. Using a collection of mutant yeast strains, we investigated various DNA metabolic and DNA repair pathways that might be involved in the maintenance of RIND-EDSB levels. We found that the RIND-EDSB levels increased significantly in yeast strains lacking proteins involved in NHEJ DNA repair and in suppression of heterochromatin formation. RIND-EDSB levels were also upregulated when genes encoding histone deacetylase, endonucleases, topoisomerase, and DNA repair regulators were deleted. In contrast, RIND-EDSB levels were downregulated in the mutants that lack chromatin-condensing proteins, such as the high-mobility group box proteins, and Sir2. Likewise, RIND-EDSB levels were also decreased in human cells lacking HMGB1. Therefore, we conclude that the genomic levels of RIND-EDSBs are evolutionally conserved, dynamically regulated, and may be influenced by genome topology, chromatin structure, and the efficiency of DNA repair systems.

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Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.

Project description:This article extends our previous quantitative analysis of the relationship between the dynamics of the primary structure of DNA and mutagenesis associated with single-strand lesions to an analysis of the production and processing of endogenous double-strand breaks (EDSBs) and to their implications for oncogenesis. We estimate that in normal human cells approximately 1% of single-strand lesions are converted to approximately 50 EDSBs per cell per cell cycle. This number is similar to that for EDSBs produced by 1.5-2.0 Gy of sparsely ionizing radiation. Although EDSBs are usually repaired with high fidelity, errors in their repair contribute significantly to the rate of cancer in humans. The doubling dose for induced DSBs is similar to doubling doses for mutation and for the induction of carcinomas by ionizing radiation. We conclude that rates of production of EDSBs and of ensuing spontaneous mitotic recombination events can account for a substantial fraction of the earliest oncogenic events in human carcinomas.

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Pathologic Replication-Independent Endogenous DNA Double-Strand Breaks Repair Defect in Chronological Aging Yeast.

Project description:Reduction of physiologic replication-independent endogenous DNA double strand breaks (Phy-RIND-EDSBs) in chronological aging yeast increases pathologic RIND-EDSBs (Path-RIND-EDSBs). Path-RIND-EDSBs can occur spontaneously in non-dividing cells without any inductive agents, and they must be repaired immediately otherwise their accumulation can lead to senescence. If yeasts have DSB repair defect, retention of Path-RIND-EDSBs can be found. Previously, we found that Path-RIND-EDSBs are not only produced but also retained in chronological aging yeast. Here, we evaluated if chronological aging yeasts have a DSB repair defect. We found a significant accumulation of Path-RIND-EDSBs around the same level in aging cells and caffeine treated cells and at a much higher level in the DSB repair mutant cells. Especially in the mutant, some unknown sequence was found inserted at the breaks. In addition, % difference of cell viability between HO induced and non-induced cells was significantly greater in aging cells. Our results suggested that RIND-EDSBs repair efficiency declines, but is not absent, in chronological aging yeast which might promote senescence phenotype. When a repair protein is deficient, an alternative pathway might be employed or an end modification process might occur as inserted sequences at the breaks were observed. Restoring repair defects might slow down the deterioration of cells from chronological aging.

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Physiological Roles of DNA Double-Strand Breaks.

Project description:Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Among the most cytotoxic lesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix. Cells boast a considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent an important category of anticancer therapeutics. And yet, bizarrely, cells deploy this very machinery for the intentional and calculated disruption of genomic integrity, harnessing potentially destructive DSBs in delicate genetic transactions. Under tight spatiotemporal regulation, DSBs serve as a tool for genetic modification, widely used across cellular biology to generate diverse functionalities, ranging from the fundamental upkeep of DNA replication, transcription, and the chromatin landscape to the diversification of immunity and the germline. Growing evidence points to a role of aberrant DSB physiology in human disease and an understanding of these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs. Here, we review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider how the cell is able to harness DNA breaks as a critical biochemical tool.

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Quantification of DNA double-strand breaks using Geant4-DNA.

Project description:PURPOSE:This study aims to standardize the simulation procedure in measuring DNA double-strand breaks (DSBs), by using advanced Monte Carlo toolkits, and newly introduced experimental methods for DNA DSB measurement. METHODS:For the experimental quantification of DNA DSB, an innovative DNA dosimeter was used to produce experimental data. GATE in combination with Geant4-DNA toolkit were exploited to simulate the experimental environment. The PDB4DNA example of Geant4-DNA was upgraded and investigated. Parameters of the simulation such energy threshold (ET) for a strand break and base pair threshold (BPT) for a DSB were evaluated, depending on the dose. RESULTS:Simulations resulted to minimum differentiation in comparison to experimental data for ET = 19 ± 1 eV and BPT = 10 bp, and high differentiation for ET<17.5 eV or ET>22.5 eV and BPT = 10 bp. There was also small differentiation for ET = 17.5 eV and BPT = 6 bp. Uncertainty has been kept lower than 3%. CONCLUSIONS:This study includes first results on the quantification of DNA double-strand breaks. The energy spectrum of a LINAC was simulated and used for the first time to irradiate DNA molecules. Simulation outcome was validated on experimental data that were produced by a prototype DNA dosimeter.

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Reading chromatin signatures after DNA double-strand breaks.

Project description:DNA double-strand breaks (DSBs) are DNA lesions that must be accurately repaired in order to preserve genomic integrity and cellular viability. The response to DSBs reshapes the local chromatin environment and is largely orchestrated by the deposition, removal and detection of a complex set of chromatin-associated post-translational modifications. In particular, the nucleosome acts as a central signalling hub and landing platform in this process by organizing the recruitment of repair and signalling factors, while at the same time coordinating repair with other DNA-based cellular processes. While current research has provided a descriptive overview of which histone marks affect DSB repair, we are only beginning to understand how these marks are interpreted to foster an efficient DSB response. Here we review how the modified chromatin surrounding DSBs is read, with a focus on the insights gleaned from structural and biochemical studies.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

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Selective mitochondrial DNA degradation following double-strand breaks.

Project description:Mitochondrial DNA (mtDNA) can undergo double-strand breaks (DSBs), caused by defective replication, or by various endogenous or exogenous sources, such as reactive oxygen species, chemotherapeutic agents or ionizing radiations. MtDNA encodes for proteins involved in ATP production, and maintenance of genome integrity following DSBs is thus of crucial importance. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown. In this study, we investigated the consequences of the production of mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSB repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content. We demonstrate that none of the known mitochondrial nucleases are involved in the mtDNA degradation and that the DNA loss is not due to autophagy, mitophagy or apoptosis. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs.

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The ubiquitin landscape at DNA double-strand breaks.

Project description:The intimate relationship between DNA double-strand break (DSB) repair and cancer susceptibility has sparked profound interest in how transactions on DNA and chromatin surrounding DNA damage influence genome integrity. Recent evidence implicates a substantial commitment of the cellular DNA damage response machinery to the synthesis, recognition, and hydrolysis of ubiquitin chains at DNA damage sites. In this review, we propose that, in order to accommodate parallel processes involved in DSB repair and checkpoint signaling, DSB-associated ubiquitin structures must be nonuniform, using different linkages for distinct functional outputs. We highlight recent advances in the study of nondegradative ubiquitin signaling at DSBs, and discuss how recognition of different ubiquitin structures may influence DNA damage responses.

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Ku recruits XLF to DNA double-strand breaks.

Project description:XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.

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