RESUMEN
Ionizing radiation (IR) is environmentally prevalent and, depending on dose and linear energy transfer (LET), can elicit serious health effects by damaging DNA. Relative to low LET photon radiation (X-rays, gamma rays), higher LET particle radiation produces more disease causing, complex DNA damage that is substantially more challenging to resolve quickly or accurately. Despite the majority of human lifetime IR exposure involving long-term, repetitive, low doses of high LET alpha particles (e.g. radon gas inhalation), technological limitations to deliver alpha particles in the laboratory conveniently, repeatedly, over a prolonged period, in low doses and in an affordable, high-throughput manner have constrained DNA damage and repair research on this topic. To resolve this, we developed an inexpensive, high capacity, 96-well plate-compatible alpha particle irradiator capable of delivering adjustable, low mGy/s particle radiation doses in multiple model systems and on the benchtop of a standard laboratory. The system enables monitoring alpha particle effects on DNA damage repair and signalling, genome stability pathways, oxidative stress, cell cycle phase distribution, cell viability and clonogenic survival using numerous microscopy-based and physical techniques. Most importantly, this method is foundational for high-throughput genetic screening and small molecule testing in mammalian and yeast cells.
Asunto(s)
Partículas alfa/efectos adversos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Genética de Radiación/instrumentación , Células A549 , Ciclo Celular/efectos de la radiación , Células HeLa , Humanos , Estrés Oxidativo/efectos de la radiación , Saccharomyces cerevisiae , Transducción de Señal/efectos de la radiaciónRESUMEN
Quantification in proteomics largely relies on the incorporation of stable isotopes, with protocols that either introduce the label through metabolic incorporation or chemical tagging. Most methods rely on the use of trypsin and/or LysC to generate labeled peptides. Although alternative proteases can enhance proteome coverage, generic quantitative methods that port over to such enzymes are lacking. Here we describe a quantification strategy amenable to most proteases, which involves propionylation of metabolically labeled lysine, using a "silent stable isotope labeling by amino acids in cell culture (SILAC)" strategy that reveals isotopic labels on second-stage mass spectrometry (MS2) fragmentation in a tandem mass tag (TMT)-like manner. We selectively propionylated lysine residues prior to digestion to generate pure ArgC-like digestion for trypsin and novel ArgN-like digestions for LysargiNase, by restricting digestion at lysine. The modification offers highly complementary sequence coverage, and even enhanced protein identification rates in certain situations (GluC digestion). Propionylated lysine residues were present in the majority of identified peptides generated from digests of cell lysates and led to the consistent release of an intense cyclic imine reporter ion at mass-to-charge ratio ( m/ z) 140 using higher-energy collisional dissociation. We grew A549 cells in media containing either l-1-13C-lysine or l-6-13C-lysine, to generate proteins that share the same accurate mass when paired. Peptides were indistinguishable on the first-stage mass spectrometry (MS1) level and, upon fragmentation, released reporter ions at m/ z 140 and m/ z 141, without otherwise affecting sequence ion mass. The quantification approach is independent of the number of peptide lysines and offers a new strategy for quantitative proteomics.
Asunto(s)
Anhídridos/análisis , Lisina/análisis , Fragmentos de Péptidos/análisis , Propionatos/análisis , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Células A549 , Animales , Bovinos , Técnicas de Cultivo de Célula , Células HeLa , Caballos , Humanos , Marcaje Isotópico/métodos , Péptido Hidrolasas/química , Proteínas/análisis , Proteolisis , Tripsina/químicaRESUMEN
Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3ß complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3ß or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3ß-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.
Asunto(s)
Daño del ADN , Fosfoproteínas Fosfatasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Represoras/metabolismo , División Celular , ADN/efectos de la radiación , Fase G2 , Células HeLa , Humanos , Modelos Biológicos , Fosforilación , Radiación Ionizante , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Heterocromatina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , Desoxirribonucleasas/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Heterocromatina/efectos de la radiación , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Ratones , Células 3T3 NIH , Radiación Ionizante , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de la radiación , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Cellular senescence is a state of permanent replicative arrest that allows cells to stay viable and metabolically active but resistant to apoptotic and mitogenic stimuli. Specific, validated markers can identify senescent cells, including senescence-associated ß galactosidase activity, chromatin alterations, cell morphology changes, activated p16- and p53-dependent signaling and permanent cell cycle arrest. Senescence is a natural consequence of DNA replication-associated telomere erosion, but can also be induced prematurely by telomere-independent events such as failure to repair DNA double strand breaks. Here, we review the molecular pathways of senescence onset, focussing on the changes in chromatin organization that are associated with cellular senescence, particularly senescence-associated heterochromatin foci formation. We also discuss the altered dynamics of the DNA double strand break response within the context of aging cells. Appreciating how, mechanistically, cellular senescence is induced, and how changes to chromatin organization and DNA repair contributes to this, is fundamental to our understanding of the normal and premature human aging processes associated with loss of organ and tissue function in humans.
Asunto(s)
Senescencia Celular/genética , Cromatina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Animales , HumanosRESUMEN
Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Reparación del ADN por Recombinación , Animales , Proteína BRCA1/antagonistas & inhibidores , Línea Celular Tumoral , Células Cultivadas , Roturas del ADN de Doble Cadena , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Fase G2/genética , Heterocromatina/metabolismo , Humanos , Ratones , Proteínas Represoras/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
The Mre11/Rad50/Nbs1 (MRN) complex has a central function in facilitating activation of the ATM protein kinase at sites of DNA double-strand breaks (DSBs). However, several other factors are also required in human cells for efficient signalling through MRN and ATM, including the tumour suppressor proteins p53-binding protein 1 (53BP1) and BRCA1. In this study, we investigate the functions of these mediator proteins in ATM activation and find that the presence of 53BP1 and BRCA1 can amplify the effects of MRN when interactions between MRN and ATM are compromised. This effect is dependent on a direct interaction between MRN and the tandem breast cancer carboxy-terminal (BRCT) repeats in 53BP1, and is accompanied by hyper-phosphorylation of both Nbs1 and 53BP1. We also find that the BRCT domains of 53BP1 affect the overall structure of 53BP1 multimers and that this structure is important for promoting ATM phosphorylation of substrates as well as for the repair of DNA DSBs in mammalian cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/fisiología , Activación Enzimática/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Homóloga de MRE11 , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Fosforilación , Unión Proteica/genética , Multimerización de Proteína/genética , Proteínas Serina-Treonina Quinasas/fisiología , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Repetitive, long-term inhalation of radioactive radon gas is one of the leading causes of lung cancer, with exposure differences being a function of geographic location, built environment, personal demographics, activity patterns, and decision-making. Here, we examine radon exposure disparities across the urban-to-rural landscape, based on 42,051 Canadian residential properties in 2034 distinct communities. People living in rural, lower population density communities experience as much as 31.2% greater average residential radon levels relative to urban equivalents, equating to an additional 26.7 Bq/m3 excess in geometric mean indoor air radon, and an additional 1 mSv/year in excess alpha radiation exposure dose rate to the lungs for occupants. Pairwise and multivariate analyses indicate that community-based radon exposure disparities are, in part, explained by increased prevalence of larger floorplan bungalows in rural areas, but that a majority of the effect is attributed to proximity to, but not water use from, drilled groundwater wells. We propose that unintended radon gas migration in the annulus of drilled groundwater wells provides radon migration pathways from the deeper subsurface into near-surface materials. Our findings highlight a previously under-appreciated determinant of radon-induced lung cancer risk, and support a need for targeted radon testing and reduction in rural communities.
Asunto(s)
Contaminantes Radiactivos del Aire , Contaminación del Aire Interior , Agua Subterránea , Neoplasias Pulmonares , Monitoreo de Radiación , Radón , Humanos , Radón/efectos adversos , Radón/análisis , Contaminantes Radiactivos del Aire/análisis , Contaminación del Aire Interior/análisis , Población Rural , Vivienda , Canadá , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/etiologíaRESUMEN
Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of approximately 15% of X- or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Fase G2/efectos de la radiación , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , ADN Helicasas , Reparación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas , Fibroblastos/efectos de la radiación , Fase G1/efectos de la radiación , Eliminación de Gen , Células HeLa , Heterocromatina/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The protein and DNA complex known as chromatin is a dynamic structure, adapting to alter the spatial arrangement of genetic information within the nucleus to meet the ever changing demands of life. Following decades of research, a dizzying array of regulatory factors is now known to control the architecture of chromatin at nearly every level. Amongst these, ATP-dependent chromatin remodelling enzymes play a key role, required for the establishment, maintenance and re-organization of chromatin through their ability to adjust the contact points between DNA and histones, the spacing between individual nucleosomes and the over-arching chromatin superstructure. Utilizing energy from ATP hydrolysis, these enzymes serve as the gatekeepers of genomic access and are essential for transcriptional regulation, DNA replication and cell division. In recent years, a vital role in DNA Double Strand Break (DSB) repair has emerged, particularly within complex chromatin environments such as heterochromatin, or regions undergoing energetic transactions such as transcription or DNA replication. Here, we will provide an overview of what is understood about ATP-dependent chromatin remodelling enzymes in the context of the DNA damage response. We will first touch upon all four major chromatin remodelling enzyme families and then focus chiefly on the nine members of the Chromodomain, Helicase, DNA-binding (CHD) family, particularly CHD3, CHD4, CHD5 and CHD6. These four proteins have established and emerging roles in DNA repair, the oxidative stress response, the maintenance of genomic stability and/or cancer prevention.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Daño del ADN/genética , ADN Helicasas/fisiología , Proteínas de Unión al ADN/fisiología , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Humanos , Modelos BiológicosRESUMEN
BACKGROUND: Alpha (α)-radiation is a ubiquitous environmental agent with epigenotoxic effects. Human exposure to α-radiation at potentially harmful levels can occur repetitively over the long term via inhalation of naturally occurring radon gas that accumulates in enclosed spaces, or as a result of a single exposure from a nuclear accident. Alterations in epigenetic DNA methylation (DNAm) have been implicated in normal aging and cancer pathogenesis. Nevertheless, the effects of aberrations in the methylome of human lung cells following exposure to single or multiple α-irradiation events on these processes remain unexplored. RESULTS: We performed genome-wide DNAm profiling of human embryonic lung fibroblasts from control and irradiated cells using americium-241 α-sources. Cells were α-irradiated in quadruplicates to seven doses using two exposure regimens, a single-fraction (SF) where the total dose was given at once, and a multi-fraction (MF) method, where the total dose was equally distributed over 14 consecutive days. Our results revealed that SF irradiations were prone to a decrease in DNAm levels, while MF irradiations mostly increased DNAm. The analysis also showed that the gene body (i.e., exons and introns) was the region most altered by both the SF hypomethylation and the MF hypermethylation. Additionally, the MF irradiations induced the highest number of differentially methylated regions in genes associated with DNAm biomarkers of aging, carcinogenesis, and cardiovascular disease. The DNAm profile of the oncogenes and tumor suppressor genes suggests that the fibroblasts manifested a defensive response to the MF α-irradiation. Key DNAm events of ionizing radiation exposure, including changes in methylation levels in mitochondria dysfunction-related genes, were mainly identified in the MF groups. However, these alterations were under-represented, indicating that the mitochondria undergo adaptive mechanisms, aside from DNAm, in response to radiation-induced oxidative stress. CONCLUSIONS: We identified a contrasting methylomic profile in the lung fibroblasts α-irradiated to SF compared with MF exposures. These findings demonstrate that the methylome response of the lung cells to α-radiation is highly dependent on both the total dose and the exposure regimen. They also provide novel insights into potential biomarkers of α-radiation, which may contribute to the development of innovative approaches to detect, prevent, and treat α-particle-related diseases.
Asunto(s)
Metilación de ADN , ADN , Humanos , Fibroblastos , Pulmón , BiomarcadoresRESUMEN
The COVID-19 pandemic has produced widespread behaviour changes that shifted how people split their time between different environments, altering health risks. Here, we report an update of North American activity patterns before and after pandemic onset, and implications to radioactive radon gas exposure, a leading cause of lung cancer. We surveyed 4009 Canadian households home to people of varied age, gender, employment, community, and income. Whilst overall time spent indoors remained unchanged, time in primary residence increased from 66.4 to 77% of life (+ 1062 h/y) after pandemic onset, increasing annual radiation doses from residential radon by 19.2% (0.97 mSv/y). Disproportionately greater changes were experienced by younger people in newer urban or suburban properties with more occupants, and/or those employed in managerial, administrative, or professional roles excluding medicine. Microinfluencer-based public health messaging stimulated health-seeking behaviour amongst highly impacted, younger groups by > 50%. This work supports re-evaluating environmental health risks modified by still-changing activity patterns.
Asunto(s)
Contaminantes Radiactivos del Aire , Contaminación del Aire Interior , COVID-19 , Neoplasias Pulmonares , Radón , Humanos , Pandemias , Contaminación del Aire Interior/efectos adversos , Contaminación del Aire Interior/análisis , Canadá/epidemiología , Exposición a Riesgos Ambientales/efectos adversos , Exposición a Riesgos Ambientales/análisis , Medición de Riesgo , COVID-19/epidemiología , COVID-19/complicaciones , Radón/toxicidad , Radón/análisis , Contaminantes Radiactivos del Aire/análisis , Neoplasias Pulmonares/epidemiología , GasesRESUMEN
It has long been known that the level of radiosensitivity between individuals covers a considerable range. This range is reflected in analysis of patient cell lines with some cell lines showing significantly reduced sensitivity to in vitro radiation exposure. Our increased exposure to radiation from diagnostic medical procedures and other life style changes has raised concerns that there may be individuals who are at an elevated risk from the harmful impact of acute or chronic low dose radiation exposure. Additionally, a subset of patients show an enhanced normal tissue response following radiotherapy, which can cause significant discomfort and, at the extreme, be life threatening. It has long been realised that the ability to identify sensitive individuals and to understand the mechanistic basis underlying the range of sensitivity within the population is important. A reduced ability to efficiently repair DNA double strand breaks (DSB) and/or activate the DSB damage response underlies some, although not necessarily all, of this sensitivity. In this article, we consider the utility of the recently developed γH2AX foci analysis to provide insight into radiation sensitivity within the population. We consider the nature of sensitivity including the impact of radiation on cell survival, tissue responses and carcinogenesis and the range of responses within the population. We overview the current utility of the γH2AX assay for assessing the efficacy of the DNA damage response to low and high dose radiation and its potential future exploitation.
Asunto(s)
Biomarcadores/análisis , Tolerancia a Radiación/genética , Línea Celular , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Histonas/análisis , HumanosRESUMEN
Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB) has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC) and highly compacted, transcriptionally inert, heterochromatin (HC), although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs) are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM), is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/fisiología , Heterocromatina/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Eucromatina/genética , Eucromatina/metabolismo , Heterocromatina/genética , HumanosRESUMEN
Exposure to environmental ionizing radiation is prevalent, with greatest lifetime doses typically from high Linear Energy Transfer (high-LET) alpha particles via the radioactive decay of radon gas in indoor air. Particle radiation is highly genotoxic, inducing DNA damage including oxidative base lesions and DNA double strand breaks. Due to the ionization density of high-LET radiation, the consequent damage is highly clustered wherein ≥2 distinct DNA lesions occur within 1-2 helical turns of one another. These multiply-damaged sites are difficult for eukaryotic cells to resolve either quickly or accurately, resulting in the persistence of DNA damage and/or the accumulation of mutations at a greater rate per absorbed dose, relative to lower LET radiation types. The proximity of the same and different types of DNA lesions to one another is challenging for DNA repair processes, with diverse pathways often confounding or interplaying with one another in complex ways. In this context, understanding the state of the higher order chromatin compaction and arrangements is essential, as it influences the density of damage produced by high-LET radiation and regulates the recruitment and activity of DNA repair factors. This review will summarize the latest research exploring the processes by which clustered DNA damage sites are induced, detected, and repaired in the context of chromatin.
RESUMEN
Radioactive radon gas inhalation causes lung cancer, and public health strategies have responded by promoting testing and exposure reduction by individuals. However, a better understanding of how radon exposure disparities are driven by psychological and social variables is required. Here, we explored how behavioural factors modified residential radon-related radiation doses incurred by 2390 people who performed a radon test. The average time from first awareness to receiving a radon test outcome was 6.8-25.5 months, depending on behaviour and attitudes. 20.5% displayed radon test urgency that reduced irradiation between awareness and outcome to 1.8 mSv from a typical 3.5 mSv, while 14.8% (more likely to be men) displayed delaying behaviours that increased exposure to 8.0 mSv. Of those with low radon, 45.9% indicated no future testing intention, underscoring the importance of original tests to reliably establish risk. Among people finding high radon, 38% mitigated quickly, 29% reported economic impediments, and 33% displayed delaying behaviours. Economic barriers and delaying behaviours resulted in 8.4 mSv/year or 10.3 mSv/year long term excess exposure, respectively, increasing lifetime risk of lung cancer by ~ 30-40%. Excess radiation doses incurred from behaviour were independent of household radon level, highlighting the strong influence of psychological and socioeconomic factors on radon exposure and lung cancer risks.
Asunto(s)
Neoplasias Pulmonares , Exposición a la Radiación , Radón , Femenino , Humanos , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/etiología , Masculino , Exposición a la Radiación/efectos adversos , Radón/análisis , Radón/toxicidad , Factores SocialesRESUMEN
Cranial irradiation is part of the standard of care for treating pediatric brain tumors. However, ionizing radiation can trigger serious long-term neurologic sequelae, including oligodendrocyte and brain white matter loss enabling neurocognitive decline in children surviving brain cancer. Oxidative stress-mediated oligodendrocyte precursor cell (OPC) radiosensitivity has been proposed as a possible explanation for this. Here, however, we demonstrate that antioxidants fail to improve OPC viability after irradiation, despite suppressing oxidative stress, suggesting an alternative etiology for OPC radiosensitivity. Using systematic approaches, we find that OPCs have higher irradiation-induced and endogenous γH2AX foci compared to neural stem cells, neurons, astrocytes and mature oligodendrocytes, and these correlate with replication-associated DNA double strand breakage. Furthermore, OPCs are reliant upon ATR kinase and Mre11 nuclease-dependent processes for viability, are more sensitive to drugs increasing replication fork collapse, and display synthetic lethality with PARP inhibitors after irradiation. This suggests an insufficiency for homology-mediated DNA repair in OPCs-a model that is supported by evidence of normal RPA but reduced RAD51 filament formation at resected lesions in irradiated OPCs. We therefore propose a DNA repair-centric mechanism of OPC radiosensitivity, involving chronically-elevated replication stress combined with 'bottlenecks' in RAD51-dependent DNA repair that together reduce radiation resilience.
RESUMEN
XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Ligasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Células Cultivadas , Cricetinae , ADN Ligasa (ATP) , Etopósido/toxicidad , Humanos , Ratones , Cinostatina/toxicidadRESUMEN
Radioactive radon gas inhalation is a major cause of lung cancer worldwide and is a consequence of the built environment. The average radon level of properties built in a given period (their 'innate radon risk') varies over time and by region, although the underlying reasons for these differences are unclear. To investigate this, we analyzed long term radon tests and buildings from 25,489 Canadian to 38,596 Swedish residential properties constructed after 1945. While Canadian and Swedish properties built from 1970 to 1980s are comparable (96-103 Bq/m3), innate radon risks subsequently diverge, rising in Canada and falling in Sweden such that Canadian houses built in the 2010-2020s have 467% greater radon (131 Bq/m3) versus Swedish equivalents (28 Bq/m3). These trends are consistent across distinct building types, and regional subdivisions. The introduction of energy efficiency measures (such as heat recovery ventilation) within each nation's build codes are independent of radon fluctuations over time. Deep learning-based models forecast that (without intervention) the average Canadian residential radon level will increase to 176 Bq/m3 by 2050. Provisions in the 2010 Canada Build Code have not significantly reduced innate radon risks, highlighting the urgency of novel code interventions to achieve systemic radon reduction and cancer prevention in Canada.
RESUMEN
Radioactive radon inhalation is a leading cause of lung cancer and underlies an ongoing public health crisis. Radon exposure prevention strategies typically begin by informing populations about health effects, and their initial efficacy is measured by how well and how fast information convinces individuals to test properties. This communication process is rarely individualized, and there is little understanding if messages impact diverse demographics equally. Here, we explored how 2,390 people interested in radon testing differed in their reaction to radon's public health information and their subsequent decision to test. Only 20% were prompted to radon test after 1 encounter with awareness information, while 65% required 2-5 encounters over several months, and 15% needed 6 to > 10 encounters over many years. People who most delayed testing were more likely to be men or involved in engineering, architecture, real estate and/or physical science-related professions. Social pressures were not a major factor influencing radon testing. People who were the least worried about radon health risks were older and/or men, while negative emotional responses to awareness information were reported more by younger people, women and/or parents. This highlights the importance of developing targeted demographic messaging to create effective radon exposure prevention strategies.