RESUMEN
BACKGROUND/AIMS: One of the treatments for breast cancer is surgical resection of the tumour and prevention of recurrence with postoperative radiotherapy. Unfortunately, radiotherapy is not always effective enough due to the low sensitivity of cancer cells to ionising radiation. This study aimed to evaluate the radiosensitising properties of resveratrol, piceatannol and polydatin on breast cancer cells, which differ in the presence of hormonal receptors on their surface. METHODS: The experimental part was carried out on triple-negative breast cancer cells (HCC38) and hormone-dependent cells (MCF7). The study assessed the level of cell death, changes in the expression of genes (BAX, BCL-2) and proteins related to the apoptosis process (CASPASE 3, 8 and P53), changes in the expression of antioxidant enzymes (CATALASE, SOD, GPx1/2) and NRF-2. Additionally, the expression level of RAD51 protein and histone H2AX, which are involved in DNA repair processes, was assessed. Statistical significance was evaluated by a two-way analysis of variance (ANOVA) followed by Tukey's post hoc test (p < 0.05). RESULTS: Ionising radiation in combination with resveratrol or piceatannol activates the apoptosis process by internal and external pathways. Greater sensitivity of MCF7 cells compared to HCC38 cells to ionising radiation in combination with resveratrol is associated with a weaker antioxidant response of cells and reduced intensity of DNA damage repair. DNA repair induced by ionising radiation occurs more effectively in HCC38 cells than in MCF7 cells. CONCLUSION: Resveratrol has the highest radiosensitising potential among the tested stilbene for cells of both lines. The effectiveness of ionizing radiation in combination with resveratrol (to a lesser extent with piceatannol) is more significant in MCF7 than in HCC38 cells.
Asunto(s)
Apoptosis , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones , Resveratrol , Estilbenos , Humanos , Estilbenos/farmacología , Resveratrol/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Femenino , Fármacos Sensibilizantes a Radiaciones/farmacología , Línea Celular Tumoral , Células MCF-7 , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/radioterapia , Neoplasias de la Mama/tratamiento farmacológico , Histonas/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Recombinasa Rad51/metabolismo , Caspasa 3/metabolismo , GlucósidosRESUMEN
Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52's ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.
Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Estructuras R-Loop , Proteína Recombinante y Reparadora de ADN Rad52 , Transcripción Genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Replicación del ADN/genética , Estructuras R-Loop/genética , Humanos , Daño del ADN , Roturas del ADN de Doble Cadena , ADN/metabolismo , ADN/genética , Reparación del ADN , Mutación , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
BACKGROUND: The histone variant macroH2A (mH2A), the most deviant variant, is about threefold larger than the conventional histone H2A and consists of a histone H2A-like domain fused to a large Non-Histone Region responsible for recruiting PARP-1 to chromatin. The available data suggest that the histone variant mH2A participates in the regulation of transcription, maintenance of heterochromatin, NAD+ metabolism, and double-strand DNA repair. RESULTS: Here, we describe a novel function of mH2A, namely its implication in DNA oxidative damage repair through PARP-1. The depletion of mH2A affected both repair and cell survival after the induction of oxidative lesions in DNA. PARP-1 formed a specific complex with mH2A nucleosomes in vivo. The mH2A nucleosome-associated PARP-1 is inactive. Upon oxidative damage, mH2A is ubiquitinated, PARP-1 is released from the mH2A nucleosomal complex, and is activated. The in vivo-induced ubiquitination of mH2A, in the absence of any oxidative damage, was sufficient for the release of PARP-1. However, no release of PARP-1 was observed upon treatment of the cells with either the DNA alkylating agent MMS or doxorubicin. CONCLUSIONS: Our data identify a novel pathway for the repair of DNA oxidative lesions, requiring the ubiquitination of mH2A for the release of PARP-1 from chromatin and its activation.
Asunto(s)
Daño del ADN , Reparación del ADN , Histonas , Poli(ADP-Ribosa) Polimerasa-1 , Ubiquitinación , Histonas/metabolismo , Histonas/genética , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Estrés Oxidativo , Nucleosomas/metabolismoRESUMEN
Utilizing molecular dynamics and free energy perturbation, we examine the relative binding affinity of several covalent polycyclic aromatic hydrocarbon - DNA (PAH-DNA) adducts at the central adenine of NRAS codon-61, a mutational hotspot implicated in cancer risk. Several PAHs classified by the International Agency for Research on Cancer as probable, possible, or unclassifiable as to carcinogenicity are found to have greater binding affinity than the known carcinogen, benzo[a]pyrene (B[a]P). van der Waals interactions between the intercalated PAH and neighboring nucleobases, and minimal disruption of the DNA duplex drive increases in binding affinity. PAH-DNA adducts may be repaired by global genomic nucleotide excision repair (GG-NER), hence we also compute relative free energies of complexation of PAH-DNA adducts with RAD4-RAD23 (the yeast ortholog of human XPC-RAD23) which constitutes the recognition step in GG-NER. PAH-DNA adducts exhibiting the greatest DNA binding affinity also exhibit the least RAD4-RAD23 complexation affinity and are thus predicted to resist the GG-NER machinery, contributing to their genotoxic potential. In particular, the fjord region PAHs dibenzo[a,l]pyrene, benzo[g]chrysene, and benzo[c]phenanthrene are found to have greater binding affinity while having weaker RAD4-RAD23 complexation affinity than their respective bay region analogs B[a]P, chrysene, and phenanthrene. We also find that the bay region PAHs dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, and dibenzo[a,h]anthracene exhibit greater binding affinity and weaker RAD4-RAD23 complexation affinity than B[a]P. Thus, the study of PAH genotoxicity likely needs to be substantially broadened, with implications for public policy and the health sciences. This approach can be broadly applied to assess factors contributing to the genotoxicity of other unclassified compounds.
Asunto(s)
Aductos de ADN , Hidrocarburos Policíclicos Aromáticos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , Aductos de ADN/química , Aductos de ADN/metabolismo , Aductos de ADN/genética , Humanos , Reparación del ADN , Mutágenos/toxicidad , Mutágenos/química , Simulación de Dinámica Molecular , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Termodinámica , Benzo(a)pireno/toxicidad , Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , ADN/química , ADN/metabolismo , Benzopirenos/toxicidad , Benzopirenos/química , Benzopirenos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/químicaRESUMEN
BACKGROUND: Atherosclerosis, serving as the primary pathological mechanism at the core of cardiovascular disease, is now widely acknowledged to be associated with DNA damage and repair, contributing to atherosclerotic plaque formation. Therefore, molecules involved in the DNA repair process may play an important role in the progression of atherosclerosis. Our research endeavors to explore the contributions of specific and interrelated molecules involved in DNA repair (APE1, BRCA1, ERCC2, miR-221-3p, miR-145-5p, and miR-155-5p) to the development of atherosclerotic plaque and their interactions with each other. METHODS & RESULTS: Gene expression study was conducted using the real-time polymerase chain reaction (qRT-PCR) method on samples from carotid artery atherosclerotic plaques and nonatherosclerotic internal mammary arteries obtained from 50 patients diagnosed with coronary artery disease and carotid artery disease. Additionally, 50 healthy controls were included for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Although no difference was observed in mRNA gene expressions, we noted a decrease in miR-155-5p gene expression (p = 0.003) and an increase in miR-221-3p gene expression (p = 0.015) in plaque samples, while miR-145-5p gene expression remained unchanged (p = 0.57). Regarding serum 8-OHdG levels, patients exhibited significantly higher levels (1111.82 ± 28.64) compared to controls (636.23 ± 24.23) (p < 0.0001). CONCLUSIONS: In our study demonstrating the role of miR-155-5p and miR-221-3p in atherosclerosis, we propose that these molecules are potential biomarkers and therapeutic targets for coronary artery diseases and carotid artery disease.
Asunto(s)
Reparación del ADN , MicroARNs , Placa Aterosclerótica , Humanos , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Femenino , Masculino , Persona de Mediana Edad , Reparación del ADN/genética , MicroARNs/genética , MicroARNs/metabolismo , Anciano , Estudios Transversales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Daño del ADN/genética , Regulación de la Expresión Génica/genética , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , 8-Hidroxi-2'-Desoxicoguanosina/metabolismoRESUMEN
BACKGROUND: Premature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear. METHODS: We measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose. RESULTS: USP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo. CONCLUSIONS: Upregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.
Asunto(s)
Senescencia Celular , Daño del ADN , Reparación del ADN , Células de la Granulosa , Insuficiencia Ovárica Primaria , Ubiquitina Tiolesterasa , Femenino , Insuficiencia Ovárica Primaria/patología , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/patología , Senescencia Celular/efectos de los fármacos , Animales , Humanos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Reparación del ADN/efectos de los fármacos , Ratones , Adulto , Ratones Endogámicos C57BL , Línea CelularRESUMEN
The formation of nuclear biomolecular condensates is often associated with local accumulation of proteins at a site of DNA damage. The key role in the formation of DNA repair foci belongs to PARP1, which is a sensor of DNA damage and catalyzes the synthesis of poly(ADP-ribose) attracting repair factors. We show here that biogenic cations such as Mg2+, Ca2+, Mn2+, spermidine3+, or spermine4+ can induce liquid-like assembly of poly(ADP-ribosyl)ated [PARylated] PARP1 into multimolecular associates (hereafter: self-assembly). The self-assembly of PARylated PARP1 affects the level of its automodification and hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG). Furthermore, association of PARylated PARP1 with repair proteins strongly stimulates strand displacement DNA synthesis by DNA polymerase ß (Pol ß) but has no noticeable effect on DNA ligase III activity. Thus, liquid-like self-assembly of PARylated PARP1 may play a critical part in the regulation of i) its own activity, ii) PARG-dependent hydrolysis of poly(ADP-ribose), and iii) Pol ß-mediated DNA synthesis. The latter can be considered an additional factor influencing the choice between long-patch and short-patch DNA synthesis during repair.
Asunto(s)
Poli(ADP-Ribosa) Polimerasa-1 , Poli ADP Ribosilación , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Humanos , Poli Adenosina Difosfato Ribosa/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Cationes Bivalentes/metabolismo , Reparación del ADN , ADN Polimerasa beta/metabolismo , Cationes/metabolismo , Daño del ADNRESUMEN
Cellular oxidative stress mediated by intrinsic and/or extrinsic reactive oxygen species (ROS) is associated with disease pathogenesis. Oxidative DNA damage can naturally be substituted by mitochondrial DNA (mtDNA), leading to base lesion/strand break formation, copy number changes, and mutations. In this study, we devised a single test for the sensitive quantification of acute mtDNA damage, repair, and copy number changes using supercoiling-sensitive quantitative PCR (ss-qPCR) and examined how oxidative stress-related mtDNA damage responses occur in oral cancer cells. We observed that exogenous hydrogen peroxide (H2O2) induced dynamic mtDNA damage responses, as reflected by early structural DNA damage, followed by DNA repair if damage did not exceed a particular threshold. However, high oxidative stress levels induced persistent mtDNA damage and caused a 5-30-fold depletion in mtDNA copy numbers over late responses. This dramatic depletion was associated with significant growth arrest and apoptosis, suggesting persistent functional consequences. Moreover, oral cancer cells responded differentially to oxidative injury when compared with normal cells, and different ROS species triggered different biological consequences under stress conditions. In conclusion, we developed a new method for the sensitive detection of mtDNA damage and copy number changes, with exogenous H2O2 inducing dynamic mtDNA damage responses associated with functional changes in stressed cancer cells. Finally, our method can help characterize oxidative DNA damage in cancer and other human diseases.
Asunto(s)
Daño del ADN , ADN Mitocondrial , Peróxido de Hidrógeno , Neoplasias de la Boca , Estrés Oxidativo , Especies Reactivas de Oxígeno , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Estrés Oxidativo/efectos de los fármacos , Humanos , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Peróxido de Hidrógeno/farmacología , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Reparación del ADN , Apoptosis/efectos de los fármacos , Variaciones en el Número de Copia de ADNRESUMEN
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system, and of ALS-associated C21ORF2 variants is largely unknown. Therefore, we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First, our data show C21ORF2 expression in ALS-relevant mouse and human neurons, such as spinal and cortical motor neurons. Further, the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response, mitochondria and neuronal excitability. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Mitocondrias , Neuronas Motoras , Quinasa 1 Relacionada con NIMA , Pez Cebra , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Quinasa 1 Relacionada con NIMA/genética , Quinasa 1 Relacionada con NIMA/metabolismo , Animales , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Reparación del ADN/genética , Daño del ADN , MutaciónRESUMEN
We lack the fundamental information needed to understand how DNA damage in the brain is generated and how it is controlled over a lifetime in the absence of replication check points. To address these questions, here, we integrate cell-type and region-specific features of DNA repair activity in the normal brain. The brain has the same repair proteins as other tissues, but normal, canonical repair activity is unequal and is characterized by high base excision repair (BER) and low double strand break repair (DSBR). The natural imbalance creates conditions where single strand breaks (SSBs) can convert to double strand breaks (DSBs) and reversibly switch between states in response to oxidation both in vivo and in vitro. Our data suggest that, in a normal background of repair, SSBs and DSBs are in an equilibrium which is pushed or pulled by metabolic state. Interconversion of SSB to DSBs provides a physiological check point, which would allow the formation of unrepaired DSBs for productive functions, but would also restrict them from exceeding tolerable limits.
Asunto(s)
Encéfalo , Roturas del ADN de Doble Cadena , Reparación del ADN , Animales , Ratones , Encéfalo/metabolismo , Ratones Endogámicos C57BL , Masculino , Roturas del ADN de Cadena Simple , Femenino , Reparación por EscisiónRESUMEN
Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability.
Asunto(s)
ADN Ribosómico , Inestabilidad Genómica , Recombinasa Rad51 , RecQ Helicasas , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Humanos , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Recombinación Homóloga , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Reparación del ADNRESUMEN
With the advancement of radiological medicine and nuclear industry technology, radiation is increasingly used to diagnose human health disorders. However, large-scale nuclear leakage has heightened concerns about the impact on human organs and tissues. Selenium is an essential trace element that functions in the body mainly in the form of selenoproteins. Selenium and selenoproteins can protect against radiation by stimulating antioxidant actions, DNA repair functions, and immune enhancement. While studies on reducing radiation through antioxidants have been conducted for many years, the underlying mechanisms of selenium and selenoproteins as significant antioxidants in radiation damage mitigation remain incompletely understood. Therefore, this paper aims to provide new insights into developing safe and effective radiation protection agents by summarizing the anti-radiation mechanism of selenium and selenoproteins.
Asunto(s)
Antioxidantes , Selenio , Selenoproteínas , Selenoproteínas/metabolismo , Humanos , Antioxidantes/farmacología , Tolerancia a Radiación , Reparación del ADN , Protectores contra Radiación/farmacología , AnimalesRESUMEN
53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development.
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Proteínas de la Ataxia Telangiectasia Mutada , Organoides , Proteína 1 de Unión al Supresor Tumoral P53 , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Organoides/metabolismo , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Fosforilación , Daño del ADN , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Reparación del ADN , Neurogénesis/genética , Neuronas/metabolismo , Transducción de SeñalRESUMEN
Transcription reprogramming is essential to carry out a variety of cell dynamics such as differentiation and stress response. During reprogramming of transcription, a number of adverse effects occur and potentially compromise genomic stability. Formaldehyde as an obligatory byproduct is generated in the nucleus via oxidative protein demethylation at regulatory regions, leading to the formation of DNA crosslinking damage. Elevated levels of transcription activities can result in the accumulation of unscheduled R-loop. DNA strand breaks can form if processed 5-methylcytosines are exercised by DNA glycosylase during imprint reversal. When cellular differentiation involves a large number of genes undergoing transcription reprogramming, these endogenous DNA lesions and damage-prone structures may pose a significant threat to genome stability. In this review, we discuss how DNA damage is formed during cellular differentiation, cellular mechanisms for their removal, and diseases associated with transcription reprogramming.
Asunto(s)
Reprogramación Celular , Daño del ADN , Transcripción Genética , Humanos , Animales , Reparación del ADN , Diferenciación Celular , Inestabilidad GenómicaRESUMEN
HerA is an ATP-dependent translocase that is widely distributed in archaea and some bacteria. It belongs to the HerA/FtsK translocase bacterial family, which is a subdivision of the RecA family. Currently, it is identified that HerA participates in the repair of DNA double-strand breaks (DSBs) or confers anti-phage defense by assembling other proteins into large complexes. In recent years, there has been a growing understanding of the bioinformatics, biochemistry, structure, and function of HerA subfamily members in both archaea and bacteria. This comprehensive review compares the structural disparities among diverse HerAs and elucidates their respective roles in specific life processes.
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Proteínas Bacterianas , Evolución Molecular , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Arqueales/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Archaea/metabolismo , Archaea/genética , Reparación del ADN , Roturas del ADN de Doble Cadena , Bacterias/metabolismo , Modelos MolecularesRESUMEN
Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos-the products of nucleolytic removal by Mre11-have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2-a protein known to repair DNA ends arising from abortive topoisomerase activity-on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo-contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Hidrolasas Diéster Fosfóricas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al ADN/metabolismo , ADN de Cadena Simple/metabolismo , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Reparación del ADNRESUMEN
Timely and accurate DNA replication is critical for safeguarding genome integrity and ensuring cell viability. Yet, this process is challenged by DNA damage blocking the progression of the replication machinery. To counteract replication fork stalling, evolutionary conserved DNA damage tolerance (DDT) mechanisms promote DNA damage bypass and fork movement. One of these mechanisms involves "skipping" DNA damage through repriming downstream of the lesion, leaving single-stranded DNA (ssDNA) gaps behind the advancing forks (also known as post-replicative gaps). In vertebrates, repriming in damaged leading templates is proposed to be mainly promoted by the primase and polymerase PRIMPOL. In this review, we discuss recent advances towards our understanding of the physiological and pathological conditions leading to repriming activation in human models, revealing a regulatory network of PRIMPOL activity. Upon repriming by PRIMPOL, post-replicative gaps formed can be filled-in by the DDT mechanisms translesion synthesis and template switching. We discuss novel findings on how these mechanisms are regulated and coordinated in time to promote gap filling. Finally, we discuss how defective gap filling and aberrant gap expansion by nucleases underlie the cytotoxicity associated with post-replicative gap accumulation. Our increasing knowledge of this repriming mechanism - from gap formation to gap filling - is revealing that targeting the last step of this pathway is a promising approach to exploit post-replicative gaps in anti-cancer therapeutic strategies.
Asunto(s)
Daño del ADN , ADN Primasa , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Humanos , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , Reparación del ADN , Enzimas Multifuncionales/metabolismo , ADN de Cadena Simple/metabolismoRESUMEN
Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy, the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment, we observe that RING1 contributes to the broad placement of H2AK119ub1, and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1, reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely, reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast, hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression.
Asunto(s)
Daño del ADN , Reparación del ADN , Histonas , Mutación Missense , Células-Madre Neurales , Neurogénesis , Complejo Represivo Polycomb 1 , Ubiquitinación , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/genética , Histonas/metabolismo , Histonas/genética , Neurogénesis/genética , Animales , Humanos , Células-Madre Neurales/metabolismo , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Cromatina/metabolismo , Microcefalia/genética , Microcefalia/metabolismoRESUMEN
The Turquoise Killifish is an important vertebrate for the study of aging and age-related diseases due to its short lifespan. Within Nothobranchiidae, species possess annual, semi-annual, or non-annual life-histories. We took a comparative approach and examined gene expression profiles (QuantSeq) from 62 individuals from eleven nothobranchid species that span three life-histories. Our results show significant differences in differentially expressed genes (DEGs) across life-histories with non-annuals and semi-annuals being most similar, and annuals being the most distinct. At finer scales, we recovered significant differences in DEGs for DNA repair genes and show that non-annual and semi-annuals share similar gene expression profiles, while annuals are distinct. Most of the GO terms enriched in annuals are related to metabolic processes. However, GO terms, including translation, protein transport, and DNA replication initiation also are enriched in annuals. Non-annuals are enriched in Notch signaling pathway genes and downregulated in the canonical Wnt signaling pathway compared to annual species, which suggests that non-annuals have stronger regulation in cellular processes. This study provides support for congruency in DEGs involved in these life-histories and provides strong evidence that a particular set of candidate genes may be worthy of study to investigate their role in the aging process.
Asunto(s)
Transcriptoma , Animales , Perfilación de la Expresión Génica , Ciprinodontiformes/genética , Peces Killi/genética , Reparación del ADN/genética , Estaciones del AñoRESUMEN
Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.