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1.
Phys Med ; 87: 90-98, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34130221

RESUMO

PURPOSE: Mammography is the diagnostic imaging practice used in screening to detect early lesions suspected of malignancy. It uses a low energy X-ray beam in which a low dose in the order of 2-3 mGy is delivered to patient breast cells. However, it has been speculated that it could lead to significant cell damage, when compared to conventional X-ray. We investigated the biological effects of low doses, with mean glandular doses (MGDs) of 2.5 mGy and 2.5 + 2.5 mGy, on mammary cells in vitro. METHODS: We used the non-tumorigenic cell line (MCF-10A) and two tumor cells lines (MCF-7 and MDA-MB-231). Colony formation, apoptosis, and double-strand DNA breaks (DSBs) were quantified. RESULTS: The selected MGD regimens did not alter the formation of colonies by any of the cell lines. MCF-7 cells exhibited a markedly increase in apoptosis, 24 h after the single-dose protocol; MCF-10A cells underwent apoptosis only after 72 h, with both irradiation regimens, while MDA-MB-231 cells (highly invasive and metastatic) were not susceptible to apoptosis. The detection of γH2AX histone in the nuclei of irradiated cells showed that the double-dose resulted in increase of DSBs, especially in tumor cell lines. CONCLUSIONS: Although the health benefits of early breast screening remain indisputable, our future perspective is to better understand the biological basis for the effects of low dose radiation on breast cells and to investigate if and under what conditions there would be a risky situation in repeated mammography screening, in both asymptomatic and symptomatic women.


Assuntos
Neoplasias da Mama , Mamografia , Mama , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Feminino , Humanos , Raios X
2.
Nat Commun ; 12(1): 3628, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131135

RESUMO

We present TensorSignatures, an algorithm to learn mutational signatures jointly across different variant categories and their genomic localisation and properties. The analysis of 2778 primary and 3824 metastatic cancer genomes of the PCAWG consortium and the HMF cohort shows that all signatures operate dynamically in response to genomic states. The analysis pins differential spectra of UV mutagenesis found in active and inactive chromatin to global genome nucleotide excision repair. TensorSignatures accurately characterises transcription-associated mutagenesis in 7 different cancer types. The algorithm also extracts distinct signatures of replication- and double strand break repair-driven mutagenesis by APOBEC3A and 3B with differential numbers and length of mutation clusters. Finally, TensorSignatures reproduces a signature of somatic hypermutation generating highly clustered variants at transcription start sites of active genes in lymphoid leukaemia, distinct from a general and less clustered signature of Polη-driven translesion synthesis found in a broad range of cancer types. In summary, TensorSignatures elucidates complex mutational footprints by characterising their underlying processes with respect to a multitude of genomic variables.


Assuntos
Genômica , Mutação , Neoplasias/genética , Algoritmos , Sequência de Bases , Citidina Desaminase/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Epigenômica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Modelos Genéticos , Mutagênese , Proteínas/genética , Transcrição Genética/genética
3.
Nat Commun ; 12(1): 3479, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108479

RESUMO

Human PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5'-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 in complex with an activating 5'-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD+ and histone PARylation factor 1 that changes PARP2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.


Assuntos
Dano ao DNA , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Transporte/metabolismo , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Ativação Enzimática , Humanos , NAD/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Desdobramento de Proteína
4.
Aging (Albany NY) ; 13(12): 15833-15874, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34139673

RESUMO

Cellular senescence may be a side effect of chemotherapy and other anti-cancer treatments that may promote inflammation and paracrine secondary senescence in healthy tissues. DNMT2/TRDMT1 methyltransferase is implicated in the regulation of cellular lifespan and DNA damage response (DDR). In the present study, the responses to senescence inducing concentrations of doxorubicin and etoposide in different cancer cells with DNMT2/TRDMT1 gene knockout were evaluated, namely changes in the cell cycle, apoptosis, autophagy, interleukin levels, genetic stability and DDR, and 5-mC and NSUN1-6 levels. Moreover, the effect of azacytidine post-treatment was considered. Diverse responses were revealed that was based on type of cancer cells (breast and cervical cancer, osteosarcoma and glioblastoma cells) and anti-cancer drugs. DNMT2/TRDMT1 gene knockout in drug-treated glioblastoma cells resulted in decreased number of apoptotic and senescent cells, IL-8 levels and autophagy, and increased number of necrotic cells, DNA damage and affected DDR compared to drug-treated glioblastoma cells with unmodified levels of DNMT2/TRDMT1. We suggest that DNMT2/TRDMT1 gene knockout in selected experimental settings may potentiate some adverse effects associated with chemotherapy-induced senescence.


Assuntos
Senescência Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Neoplasias/genética , Neoplasias/patologia , 5-Metilcitosina/metabolismo , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoplasma/metabolismo , Quebras de DNA de Cadeia Dupla , Técnicas de Inativação de Genes , Humanos , Estresse Oxidativo/efeitos dos fármacos , beta-Galactosidase/metabolismo , tRNA Metiltransferases/metabolismo
5.
Nat Commun ; 12(1): 4033, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188037

RESUMO

In response to DNA double-strand breaks (DSBs), repair proteins are recruited to the damaged sites. Ubiquitin signaling plays a critical role in coordinating protein recruitment during the DNA damage response. Here, we find that the microRNA biogenesis factor DGCR8 promotes tumor resistance to X-ray radiation independently of its Drosha-binding ability. Upon radiation, the kinase ATM and the deubiquitinase USP51 mediate the activation and stabilization of DGCR8 through phosphorylation and deubiquitination. Specifically, radiation-induced ATM-dependent phosphorylation of DGCR8 at serine 677 facilitates USP51 to bind, deubiquitinate, and stabilize DGCR8, which leads to the recruitment of DGCR8 and DGCR8's binding partner RNF168 to MDC1 and RNF8 at DSBs. This, in turn, promotes ubiquitination of histone H2A, repair of DSBs, and radioresistance. Altogether, these findings reveal the non-canonical function of DGCR8 in DSB repair and suggest that radiation treatment may result in therapy-induced tumor radioresistance through ATM- and USP51-mediated activation and upregulation of DGCR8.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a RNA/metabolismo , Tolerância a Radiação/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Enzimas Desubiquitinantes/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Histonas/metabolismo , Humanos , Células MCF-7 , Neoplasias/genética , Neoplasias/radioterapia , Fosforilação , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Toxicol Lett ; 349: 69-83, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34126181

RESUMO

Exposure to sterigmatocystin (STC) raises concerns on developmental neurological disorders. The present study investigated the effects of maternal oral STC exposure on postnatal hippocampal neurogenesis of offspring in rats. Dams were exposed to STC (1.7, 5.0, and 15.0 ppm in diet) from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without STC exposure until adulthood on postnatal day (PND) 77, in accordance with OECD chemical testing guideline Test No. 426. On PND 21, 15.0-ppm STC decreased type-3 neural progenitor cell numbers in the subgranular zone (SGZ) due to suppressed proliferation. Increased γ-H2AX-immunoreactive (+) cell numbers in the SGZ and Ercc1 upregulation and Brip1 downregulation in the dentate gyrus suggested induction of DNA double-strand breaks in SGZ cells. Upregulation of Apex1 and Ogg1 and downregulation of antioxidant genes downstream of NRF2-Keap1 signaling suggested induction of oxidative DNA damage. Increased p21WAF1/CIP1+ SGZ cell numbers and suppressed cholinergic signaling through CHRNB2-containing receptors in GABAergic interneurons suggested potential neurogenesis suppression mechanisms. Multiple mechanisms involving N-methyl-d-aspartate (NMDA) receptor-mediated glutamatergic signaling and various GABAergic interneuron subpopulations, including CHRNA7-expressing somatostatin+ interneurons activated by BDNF-TrkB signaling, may be involved in ameliorating the neurogenesis. Upregulation of Arc, Ptgs2, and genes encoding NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors suggested synaptic plasticity facilitation. On PND 77, ARC+ granule cells decreased, and Nos2 was upregulated following 15.0 ppm STC exposure, suggesting oxidative stress-mediated synaptic plasticity suppression. Inverse pattern in gene expression changes in vesicular glutamate transporter isoforms, Slc17a7 and Slc17a6, from weaning might also be responsible for the synaptic plasticity suppression. The no-observed-adverse-effect level of maternal oral STC exposure for offspring neurogenesis was determined to be 5.0 ppm, translating to 0.34-0.85 mg/kg body weight/day.


Assuntos
Proliferação de Células/efeitos dos fármacos , Giro Denteado/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Esterigmatocistina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Giro Denteado/metabolismo , Giro Denteado/patologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Nível de Efeito Adverso não Observado , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo , Desmame
7.
Toxicol Lett ; 349: 134-144, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34153406

RESUMO

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 µM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells.


Assuntos
Ductos Biliares/efeitos dos fármacos , Histonas/metabolismo , Macrófagos/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Propano/análogos & derivados , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Técnicas de Cocultura , Quebras de DNA de Cadeia Dupla , Humanos , Interleucina-6/metabolismo , Macrófagos/metabolismo , Propano/toxicidade , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
8.
Nat Commun ; 12(1): 3856, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158470

RESUMO

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.


Assuntos
Motivos de Aminoácidos , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
9.
Nat Commun ; 12(1): 3849, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158508

RESUMO

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Estruturas R-Loop , RNA Helicases/metabolismo , Transativadores/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HCT116 , Humanos , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Helicases/genética , Interferência de RNA , Telômero/genética , Telômero/metabolismo , Transativadores/genética
10.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071949

RESUMO

Exposing cells to DNA damaging agents, such as ionizing radiation (IR) or cytotoxic chemicals, can cause DNA double-strand breaks (DSBs), which are crucial to repair to maintain genetic integrity. O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification (PTM), which has been reported to be involved in the DNA damage response (DDR) and chromatin remodeling. Here, we investigated the impact of O-GlcNAcylation on the DDR, DSB repair and chromatin status in more detail. We also applied charged particle irradiation to analyze differences of O-GlcNAcylation and its impact on DSB repair in respect of spatial dose deposition and radiation quality. Various techniques were used, such as the γH2AX foci assay, live cell microscopy and Fluorescence Lifetime Microscopy (FLIM) to detect DSB rejoining, protein accumulation and chromatin states after treating the cells with O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) inhibitors. We confirmed that O-GlcNAcylation of MDC1 is increased upon irradiation and identified additional repair factors related to Homologous Recombination (HR), CtIP and BRCA1, which were increasingly O-GlcNAcyated upon irradiation. This is consistent with our findings that the function of HR is affected by OGT inhibition. Besides, we found that OGT and OGA activity modulate chromatin compaction states, providing a potential additional level of DNA-repair regulation.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Recombinação Homóloga , Humanos , Transferência Linear de Energia , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Radiação Ionizante
11.
Mol Cell ; 81(14): 3007-3017.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34107305

RESUMO

RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.


Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Origem de Replicação/genética , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/genética , Rad51 Recombinase/genética
12.
Neuron ; 109(11): 1766-1768, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34081919

RESUMO

Emerging evidence shows that neuronal DNA is continuously broken and repaired in a non-random fashion within the genome. Two recent studies, Wu et al. (2021) and Reid et al. (2021), use sequencing of newly synthesized DNA in post-mitotic neurons to map hotspots of DNA repair across the genome. Wu et al. (2021) further show that the repair sites are associated with single-stranded DNA breaks that predominantly occur on neuronal enhancers at sites of CpG methylation.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , DNA , Reparo do DNA , Neurônios
13.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066270

RESUMO

With the rapid growth of the wireless communication industry, humans are extensively exposed to electromagnetic fields (EMF) comprised of radiofrequency (RF). The skin is considered the primary target of EMFs given its outermost location. Recent evidence suggests that extremely low frequency (ELF)-EMF can improve the efficacy of DNA repair in human cell-lines. However, the effects of EMF-RF on DNA damage remain unknown. Here, we investigated the impact of EMF-long term evolution (LTE, 1.762 GHz, 8 W/kg) irradiation on DNA double-strand break (DSB) using the murine melanoma cell line B16 and the human keratinocyte cell line HaCaT. EMF-LTE exposure alone did not affect cell viability or induce apoptosis or necrosis. In addition, DNA DSB damage, as determined by the neutral comet assay, was not induced by EMF-LTE irradiation. Of note, EMF-LTE exposure can attenuate the DNA DSB damage induced by physical and chemical DNA damaging agents (such as ionizing radiation (IR, 10 Gy) in HaCaT and B16 cells and bleomycin (BLM, 3 µM) in HaCaT cells and a human melanoma cell line MNT-1), suggesting that EMF-LTE promotes the repair of DNA DSB damage. The protective effect of EMF-LTE against DNA damage was further confirmed by attenuation of the DNA damage marker γ-H2AX after exposure to EMF-LTE in HaCaT and B16 cells. Most importantly, irradiation of EMF-LTE (1.76 GHz, 6 W/kg, 8 h/day) on mice in vivo for 4 weeks reduced the γ-H2AX level in the skin tissue, further supporting the protective effects of EMF-LTE against DNA DSB damage. Furthermore, p53, the master tumor-suppressor gene, was commonly upregulated by EMF-LTE irradiation in B16 and HaCaT cells. This finding suggests that p53 plays a role in the protective effect of EMF-LTE against DNA DSBs. Collectively, these results demonstrated that EMF-LTE might have a protective effect against DNA DSB damage in the skin, although further studies are necessary to understand its impact on human health.


Assuntos
Quebras de DNA de Cadeia Dupla , Campos Eletromagnéticos , Queratinócitos/efeitos da radiação , Melanoma/prevenção & controle , Substâncias Protetoras , Radiação Ionizante , Ondas de Rádio , Animais , Apoptose , Sobrevivência Celular , Reparo do DNA , Humanos , Técnicas In Vitro , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Melanoma/etiologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL
14.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068283

RESUMO

DNA double-strand breaks (DSBs), interrupting the genetic information, are elicited by various environmental and endogenous factors. They bear the risk of cell lethality and, if mis-repaired, of deleterious mutation. This negative impact is contrasted by several evolutionary achievements for DSB processing that help maintaining stable inheritance (correct repair, meiotic cross-over) and even drive adaptation (immunoglobulin gene recombination), differentiation (chromatin elimination) and speciation by creating new genetic diversity via DSB mis-repair. Targeted DSBs play a role in genome editing for research, breeding and therapy purposes. Here, I survey possible causes, biological effects and evolutionary consequences of DSBs, mainly for students and outsiders.


Assuntos
Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Meiose , Animais , Edição de Genes , Humanos
15.
Cancer Sci ; 112(7): 2679-2691, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33949040

RESUMO

BCR-ABL1 gene fusion associated with additional DNA lesions involves the pathogenesis of chronic myelogenous leukemia (CML) from a chronic phase (CP) to a blast crisis of B lymphoid (CML-LBC) lineage and BCR-ABL1+ acute lymphoblastic leukemia (BCR-ABL1+ ALL). The recombination-activating gene RAG1 and RAG2 (collectively, RAG) proteins that assemble a diverse set of antigen receptor genes during lymphocyte development are abnormally expressed in CML-LBC and BCR-ABL1+ ALL. However, the direct involvement of dysregulated RAG in disease progression remains unclear. Here, we generate human wild-type (WT) RAG and catalytically inactive RAG-expressing BCR-ABL1+ and BCR-ABL1- cell lines, respectively, and demonstrate that BCR-ABL1 specifically collaborates with RAG recombinase to promote cell survival in vitro and in xenograft mice models. WT RAG-expressing BCR-ABL1+ cell lines and primary CD34+ bone marrow cells from CML-LBC samples maintain more double-strand breaks (DSB) compared to catalytically inactive RAG-expressing BCR-ABL1+ cell lines and RAG-deficient CML-CP samples, which are measured by γ-H2AX. WT RAG-expressing BCR-ABL1+ cells are biased to repair RAG-mediated DSB by the alternative non-homologous end joining pathway (a-NHEJ), which could contribute genomic instability through increasing the expression of a-NHEJ-related MRE11 and RAD50 proteins. As a result, RAG-expressing BCR-ABL1+ cells decrease sensitivity to tyrosine kinase inhibitors (TKI) by activating BCR-ABL1 signaling but independent of the levels of BCR-ABL1 expression and mutations in the BCR-ABL1 tyrosine kinase domain. These findings identify a surprising and novel role of RAG in the functional specialization of disease progression in BCR-ABL1+ leukemia through its endonuclease activity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Animais , Crise Blástica/genética , Crise Blástica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteínas de Fusão bcr-abl/genética , Instabilidade Genômica , Xenoenxertos , Histonas/análise , Proteínas de Homeodomínio/genética , Humanos , Técnicas In Vitro , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteína Homóloga a MRE11/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico
16.
Nucleic Acids Res ; 49(9): 5095-5105, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33963863

RESUMO

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.


Assuntos
Proteína 9 Associada à CRISPR , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Algoritmos , Animais , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Reação em Cadeia da Polimerase , Reparo de DNA por Recombinação
17.
Mol Cell ; 81(12): 2596-2610.e7, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33961796

RESUMO

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fase G1/fisiologia , Histonas/metabolismo , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia
18.
Mol Cell ; 81(12): 2583-2595.e6, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33961797

RESUMO

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.


Assuntos
Linhagem da Célula/genética , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Humanos , Ligação Proteica , Proteínas de Ligação a RNA/fisiologia , Domínio Tudor , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia
19.
Mol Cell Biol ; 41(7): e0008221, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33941620

RESUMO

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.


Assuntos
Transtorno Autístico/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Instabilidade Genômica/fisiologia , Proteína BRCA1 , Reparo do DNA/fisiologia , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/genética
20.
Mutat Res ; 822: 111743, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33975127

RESUMO

DNA double-strand breaks (DSBs) are one of the most dangerous assaults on the genome, and yet their natural and programmed production are inherent to life. When DSBs arise close together they are particularly deleterious, and their repair may require an altered form of the DNA damage response. Our understanding of how clustered DSBs are repaired in the germline is unknown. Using laser microirradiation, we examine early events in the repair of clustered DSBs in germ cells within Caenorhabditis elegans. We use precise temporal resolution to show how the recruitment of MRE-11 to complex damage is regulated, and that clustered DNA damage can recruit proteins from various repair pathways. Abrogation of non-homologous end joining or COM-1 attenuates the recruitment of MRE-11 through distinct mechanisms. The synaptonemal complex plays both positive and negative regulatory roles in these mutant contexts. These findings indicate that MRE-11 is regulated by modifying its accessibility to chromosomes.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cromossomos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA de Helmintos , Meiose , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , DNA de Helmintos/genética , DNA de Helmintos/metabolismo
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