Editorial: DNA damage response in the context of chromatin

All DNA transactions, including the repair of damaged DNA, take place in the context of a highly organized yet dynamic chromatin. While studies on individual proteins have yielded important insights into the constituent components of DNA repair machineries that evolved in various organisms, it has become abundantly clear that a more complete understanding of the molecular choreography of DNA repair must take into consideration the complex interplay between repair factors and the chromatin milieu in which they operate. This Research Topic collection, termed “DNA damage response in the context of chromatin”, brings together experts at the forefront of this emerging field with a series of authoritative reviews and exciting original articles to provide a timely update on our current understandings of biology at the intersection between chromatin and DNA repair

Trauma Characteristics Associated with E-Scooter Accidents in Switzerland-A Case Series Study.

E-scooters have gained popularity worldwide in the last few years. Due to the increase in users, more accidents related to e-scooters can be observed. The present study aimed to analyse epidemiological data, characteristics, and severity of injuries in patients admitted to a Level I trauma centre in Switzerland (Inselspital Bern, University Hospital Bern) after accidents associated with e-scooters. This retrospective case series evaluated 23 patients who presented to the University Hospital of Bern between 1 of May 2019 and 31 of October 2021 after an e-scooter accident. Data were collected on patient demographics, time and cause of the accident, speed, alcohol consumption, helmet use, type and localisation of injury, number of injuries per patient, and outcome. Men were most frequently affected (61.9%). The mean age was 35.8 (STD 14.8) years. Slightly more than half (52.2%) of all accidents were self-inflicted. Most accidents were reported during the night (7 p.m. to 7 a.m., 60.9%) and in summer (43.5%). Alcohol consumption was reported in 43.5% of cases, with a mean blood alcohol level of 1.4 g/l. Most injuries were observed in the face (25.3%) and head/neck area (20.25%). Skin abrasions (56.5%) and traumatic brain injury (43.5%) were the most common types of traumata in terms of total number of patients. Only in one case it was reported that a protective helmet had been worn. Five patients required hospitalisation and four patients underwent surgery. Three patients underwent emergency orthopaedic surgery, and one patient underwent emergency neurosurgery. E-scooter accidents result in a significant number of facial and head/neck injuries. E-scooter riders would potentially benefit from a helmet to protect them in the event of an accident. Additionally, the results of this study indicate that a significant number of e-scooter accidents in Switzerland occurred under the influence of alcohol. Prevention campaigns to raise awareness of the risks of driving e-scooters under the influence of alcohol could help prevent future accidents

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ∼20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ∼20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1

Oxygen minimum zone induced rapid temporal fluctuations of Eastern Baltic cod genetic diversity

Oxygen minimum zones are increasing, yet the effects of these zones on the genetic composition of marine fish stocks has been neglected. We assessed the combined effects of stock size and structure, and the prevailing oxygen situation, on Eastern Baltic cod (ICES SD25) genetic diversity. For this purpose, we used an integrative long-term otolith sample and data series (1995-2013) to (1) calculate the approximate number of females with surviving eggs in a given year, i.e., contributing to reproduction (n F), and (2) the annually resolved cohort mean allelic richness as proxy of genetic diversity, based on 12 microsatellite markers. Cohort mean allelic richness showed strong year-to-year fluctuations though no permanent decline. Importantly, it was highly correlated with n F, but with an unexpected 1.5 year time lag that may be an artefact of Eastern Baltic cod ageing problems. Our findings indicated that environmental pressure can effect rapid alterations in exploited fish stock genetic composition, and pointed to the importance of large females for Eastern Baltic cod reproduction during stagnation periods. Considering the importance of standing genetic variation for the evolutionary potential of populations, this is relevant for projections of the future state of cod stocks under global change

Induction and processing of the radiation-induced gamma-H2AX signal and Its link to the underlying pattern of DSB: A combined experimental and modelling study

We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular