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1.
Nat Commun ; 12(1): 4108, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226550

RESUMO

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.


Assuntos
DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Triagem , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Glicosilases/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Especificidade por Substrato
2.
Nat Commun ; 12(1): 4126, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226554

RESUMO

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.


Assuntos
Proteína BRCA1/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA , Recombinação Homóloga , Proteínas de Neoplasias/metabolismo , RNA , Proteína BRCA1/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , DNA Helicases , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Ftalazinas , Piperazinas , RNA Helicases , RNA Mensageiro , Rad51 Recombinase , Reparo de DNA por Recombinação
3.
Int J Mol Sci ; 22(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34281248

RESUMO

Age-related macular degeneration (AMD), the main cause of vision loss in the elderly, is associated with oxidation in the retina cells promoting telomere attrition. Activation of telomerase was reported to improve macular functions in AMD patients. The catalytic subunit of human telomerase (hTERT) may directly interact with proteins important for senescence, DNA damage response, and autophagy, which are impaired in AMD. hTERT interaction with mTORC1 (mTOR (mechanistic target of rapamycin) complex 1) and PINK1 (PTEN-induced kinase 1) activates macroautophagy and mitophagy, respectively, and removes cellular debris accumulated over AMD progression. Ectopic expression of telomerase in retinal pigment epithelium (RPE) cells lengthened telomeres, reduced senescence, and extended their lifespan. These effects provide evidence for the potential of telomerase in AMD therapy. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may be involved in AMD pathogenesis through decreasing oxidative stress and senescence, regulation of vascular endothelial growth factor (VEGF), and improving autophagy. PGC-1α and TERT form an inhibitory positive feedback loop. In conclusion, telomerase activation and its ectopic expression in RPE cells, as well as controlled clinical trials on the effects of telomerase activation in AMD patients, are justified and should be assisted by PGC-1α modulators to increase the therapeutic potential of telomerase in AMD.


Assuntos
Degeneração Macular/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Telomerase/metabolismo , Envelhecimento/metabolismo , Autofagia/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Degeneração Macular/fisiopatologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Telomerase/fisiologia , Telômero/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201700

RESUMO

DNA repair ensures genomic stability to achieve healthy ageing, including cognitive maintenance. Mutations on genes encoding key DNA repair proteins can lead to diseases with accelerated ageing phenotypes. Some of these diseases are xeroderma pigmentosum group A (XPA, caused by mutation of XPA), Cockayne syndrome group A and group B (CSA, CSB, and are caused by mutations of CSA and CSB, respectively), ataxia-telangiectasia (A-T, caused by mutation of ATM), and Werner syndrome (WS, with most cases caused by mutations in WRN). Except for WS, a common trait of the aforementioned progerias is neurodegeneration. Evidence from studies using animal models and patient tissues suggests that the associated DNA repair deficiencies lead to depletion of cellular nicotinamide adenine dinucleotide (NAD+), resulting in impaired mitophagy, accumulation of damaged mitochondria, metabolic derailment, energy deprivation, and finally leading to neuronal dysfunction and loss. Intriguingly, these features are also observed in Alzheimer's disease (AD), the most common type of dementia affecting more than 50 million individuals worldwide. Further studies on the mechanisms of the DNA repair deficient premature ageing diseases will help to unveil the mystery of ageing and may provide novel therapeutic strategies for AD.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/complicações , Dano ao DNA , Instabilidade Genômica , Doenças Neurodegenerativas/patologia , Animais , Reparo do DNA , Humanos , Mutação , Doenças Neurodegenerativas/etiologia
5.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202641

RESUMO

The cohesin complex is crucial for mediating sister chromatid cohesion and for hierarchal three-dimensional organization of the genome. Mutations in cohesin genes are present in a range of cancers. Extensive research over the last few years has shown that cohesin mutations are key events that contribute to neoplastic transformation. Cohesin is involved in a range of cellular processes; therefore, the impact of cohesin mutations in cancer is complex and can be cell context dependent. Candidate targets with therapeutic potential in cohesin mutant cells are emerging from functional studies. Here, we review emerging targets and pharmacological agents that have therapeutic potential in cohesin mutant cells.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Predisposição Genética para Doença , Mutação , Neoplasias/genética , Biomarcadores Tumorais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gerenciamento Clínico , Regulação da Expressão Gênica , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/terapia , Especificidade de Órgãos , Ligação Proteica , Relação Estrutura-Atividade
6.
Nat Commun ; 12(1): 4258, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253734

RESUMO

The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, here we show disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.


Assuntos
Autofagia , Tamanho do Núcleo Celular , Núcleo Celular/metabolismo , Dano ao DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína com Valosina/metabolismo , Animais , Biomarcadores/metabolismo , Olho Composto de Artrópodes , Reparo do DNA , Mitose , Transdução de Sinais , Fatores de Tempo , Proteínas Ubiquitinadas/metabolismo
7.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208390

RESUMO

APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein-protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Sequência de Aminoácidos , Humanos , Modelos Biológicos , Terapia de Alvo Molecular , Domínios Proteicos , Processamento de Proteína Pós-Traducional
8.
Urol Clin North Am ; 48(3): 339-347, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34210489

RESUMO

Androgen receptor function, tumor cell plasticity, loss of tumor suppressors, and defects in DNA repair genes affect aggressive features of prostate cancer. Prostate cancer development, progression, and aggressive behavior are often attributable to function of the androgen receptor. Tumor cell plasticity, neuroendocrine features, and loss of tumor suppressors lend aggressive behavior to prostate cancer cells. DNA repair defects have ramifications for prostate cancer cell behavior.


Assuntos
Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Antagonistas de Androgênios/uso terapêutico , Biomarcadores Tumorais/genética , Plasticidade Celular , Reparo do DNA , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Mutação em Linhagem Germinativa , Humanos , Masculino , Medicina de Precisão , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia
9.
Urol Clin North Am ; 48(3): 365-371, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34210491

RESUMO

Germline testing should be performed to support treatment selection for patients with metastatic prostate cancer, and should be identified in patients with high-risk localized disease. Patients with germline BRCA1/2 mutations should be educated regarding additional personal cancer risk, and risk for family members. Guidelines recommend that all men with metastatic prostate cancer should also undergo somatic tissue and germline testing for priority genes BRCA1/2, PALB2, ATM, and MSH2/6. The advent of high throughput sequencing enables patients to be tested for a more comprehensive panel of germline and somatic mutations.


Assuntos
Predisposição Genética para Doença , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Dano ao DNA , Reparo do DNA , Genes BRCA1 , Genes BRCA2 , Testes Genéticos , Mutação em Linhagem Germinativa , Humanos , Indóis/uso terapêutico , Masculino , Metástase Neoplásica/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico
10.
Anticancer Res ; 41(7): 3261-3270, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230120

RESUMO

BACKGROUND/AIM: Chronic inflammation generates large quantities of reactive oxygen and nitrogen species that damage DNA. DNA repair is important for cellular viability and genome integrity. MATERIALS AND METHODS: Expression levels of the DNA repair proteins OGG1, XPA, MLH1, PARP1, and XRCC6, which function in base excision repair, nucleotide excision repair, mismatch repair, single-strand break repair and double-strand break repair, respectively, were assessed using immunohistochemistry in ulcerative colitis and sporadic colorectal cancer biopsies. Levels of oxidative/ nitrosative stress biomarkers were also assessed. RESULTS: Ulcerative colitis and colorectal cancer lesions expressed significantly higher levels of all DNA repair proteins and oxidative/ nitrosative stress biomarkers compared to normal colonic mucosa. Ulcerative colitis had the highest XPA and XRCC6 expression. CONCLUSION: Oxidative/nitrosative stress is prevalent in the colon of both diseases. Nucleotide excision repair and non-homologous end-joining double-strand break repair may be compromised in colorectal cancer, but not in ulcerative colitis.


Assuntos
Colite Ulcerativa/genética , Neoplasias Colorretais/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Estresse Nitrosativo/genética , Estresse Oxidativo/genética , Biomarcadores Tumorais/genética , Colite Ulcerativa/patologia , Colo/patologia , Neoplasias Colorretais/patologia , Dano ao DNA/genética , Humanos , Mucosa Intestinal/patologia , Oxirredução
11.
Appl Microbiol Biotechnol ; 105(13): 5449-5460, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34223949

RESUMO

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature. Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bo nd of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave AP-containing ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. Phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs. KEY POINTS: We first reported a novel DNA glycosylase with bifunctionality. Tba UDG247 possesses an AP lyase activity.


Assuntos
Thermococcus , Reparo do DNA , Filogenia , Thermococcus/genética , Thermococcus/metabolismo , Uracila , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
12.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207557

RESUMO

Nucleotide excision repair (NER) is the most versatile DNA repair pathway, which can remove diverse bulky DNA lesions destabilizing a DNA duplex. NER defects cause several autosomal recessive genetic disorders. Xeroderma pigmentosum (XP) is one of the NER-associated syndromes characterized by low efficiency of the removal of bulky DNA adducts generated by ultraviolet radiation. XP patients have extremely high ultraviolet-light sensitivity of sun-exposed tissues, often resulting in multiple skin and eye cancers. Some XP patients develop characteristic neurodegeneration that is believed to derive from their inability to repair neuronal DNA damaged by endogenous metabolites. A specific class of oxidatively induced DNA lesions, 8,5'-cyclopurine-2'-deoxynucleosides, is considered endogenous DNA lesions mainly responsible for neurological problems in XP. Growing evidence suggests that XP is accompanied by defective mitophagy, as in primary mitochondrial disorders. Moreover, NER pathway is absent in mitochondria, implying that the mitochondrial dysfunction is secondary to nuclear NER defects. In this review, we discuss the current understanding of the NER molecular mechanism and focuses on the NER linkage with the neurological degeneration in patients with XP. We also present recent research advances regarding NER involvement in oxidative DNA lesion repair. Finally, we highlight how mitochondrial dysfunction may be associated with XP.


Assuntos
Reparo do DNA , Doenças Neurodegenerativas , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia
13.
FASEB J ; 35(8): e21753, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34233068

RESUMO

Ovarian infertility and subfertility presenting with premature ovarian insufficiency (POI) and diminished ovarian reserve are major issues facing the developed world due to the trend of delaying childbirth. Ovarian senescence and POI represent a continuum of physiological/pathophysiological changes in ovarian follicle functions. Based on advances in whole exome sequencing, evaluation of gene copy variants, together with family-based and genome-wide association studies, we discussed genes responsible for POI and ovarian senescence. We used a gene-centric approach to sort out literature deposited in the Ovarian Kaleidoscope database (http://okdb.appliedbioinfo.net) by sub-categorizing candidate genes as ligand-receptor signaling, meiosis and DNA repair, transcriptional factors, RNA metabolism, enzymes, and others. We discussed individual gene mutations found in POI patients and verification of gene functions in gene-deleted model organisms. Decreased expression of some of the POI genes could be responsible for ovarian senescence, especially those essential for DNA repair, meiosis and mitochondrial functions. We propose to set up a candidate gene panel for targeted sequencing in POI patients together with studies on mitochondria-associated genes in middle-aged subfertile patients.


Assuntos
Ovário/metabolismo , Insuficiência Ovariana Primária/genética , Animais , Reparo do DNA/genética , Bases de Dados Genéticas , Feminino , Estudo de Associação Genômica Ampla , Humanos , Meiose/genética , Menopausa Precoce/genética , Menopausa Precoce/metabolismo , Modelos Genéticos , Reserva Ovariana/genética , Insuficiência Ovariana Primária/metabolismo , Fatores de Transcrição/genética , Sequenciamento Completo do Exoma
14.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205449

RESUMO

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5',8-cyclo-2'-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction-modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme's recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5'S)-5',8-cyclo-2'-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5'R)-5',8-cyclo-2'-deoxyadenosine (RcdA). Interestingly, in the case of 5',8-cyclo-2'-deoxyguanosine (cdG), both 5'S and 5'R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.


Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Animais , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Oligonucleotídeos/metabolismo
15.
Talanta ; 232: 122429, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074415

RESUMO

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL-1 and 0.00001 U mL-1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.


Assuntos
DNA Glicosilases , Técnicas de Amplificação de Ácido Nucleico , Uracila-DNA Glicosidase , Sondas de DNA , Reparo do DNA , Células HeLa , Humanos , Limite de Detecção , Uracila-DNA Glicosidase/metabolismo
16.
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
17.
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
18.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066883

RESUMO

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.


Assuntos
Reparo do DNA/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular Tumoral , Humanos , Reprodutibilidade dos Testes
19.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070674

RESUMO

BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Predisposição Genética para Doença , Mutações Sintéticas Letais , Neoplasias da Mama/terapia , Reparo do DNA , Feminino , Humanos
20.
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
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