Borna disease virus docks on neuronal DNA double-strand breaks to replicate and dampens neuronal activity.
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ABSTRACT:
SUBMITTER: Marty FH
PROVIDER: S-EPMC8724971 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer's disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX-a post-translational histone modification that is widely used as a marker of DSBs-revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease.
| S-EPMC6524256 | biostudies-literature
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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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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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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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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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.
| S-EPMC5409072 | biostudies-literature
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.
| S-EPMC2246615 | biostudies-literature
Project description:Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and ?H2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained ?H2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as ?H2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.
| S-EPMC4871660 | biostudies-literature
Project description:DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state.
| S-EPMC4826256 | biostudies-literature
Project description:Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5' strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5' strand DNA ?10-20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5' strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP-MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.
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