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1.
Yi Chuan ; 41(9): 777-800, 2019 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-31549678

RESUMO

Base editing is a newly developed precise genome editing technique based on the CRISPR/Cas system. According to different base modification enzymes, the current base editing systems can be divided into cytosine base editors (CBE) and adenine base editors (ABE). They use cytosine deaminases or artificially evolved adenine deaminases to perform single-base editing, and achieve C to T (G to A) or A to G (T to C) substitutions, respectively. Due to high efficiency, independence of DNA double-strand breaks, and no need for donor DNA, base editing systems have been successfully applied in diverse species including animals, plants and other organisms since the first report in 2016. Therefore, base editing systems will have a high prospect of providing important support for gene therapy and crop genetic improvement in the future. In this review, we describe the development and current applications of base editing systems for basic research and biotechnology, highlight the challenges, and discuss the directions for future research in this important field. The information presented may facilitate interested researchers to grasp the principles of base editing, to use relevant base editing tools in their own studies, or to innovate new versions of base editing in the future.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Edição de Genes , Adenina , Aminoidrolases , Animais , Biotecnologia/tendências , Citosina , Citosina Desaminase , Plantas
2.
Adv Exp Med Biol ; 1152: 9-29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456177

RESUMO

Epidemiologic studies have contributed importantly to current knowledge of environmental and genetic risk factors for breast cancer. Worldwide, breast cancer is an important cause of human suffering and premature mortality among women. In the United States, breast cancer accounts for more cancer deaths in women than any site other than lung cancer. A variety of risk factors for breast cancer have been well-established by epidemiologic studies including race, ethnicity, family history of cancer, and genetic traits, as well as modifiable exposures such as increased alcohol consumption, physical inactivity, exogenous hormones, and certain female reproductive factors. Younger age at menarche, parity, and older age at first full-term pregnancy may influence breast cancer risk through long-term effects on sex hormone levels or by other biological mechanisms. Recent studies have suggested that triple negative breast cancers may have a distinct etiology. Genetic variants and mutations in genes that code for proteins having a role in DNA repair pathways and the homologous recombination of DNA double stranded breaks (APEX1, BRCA1, BRCA2, XRCC2, XRCC3, ATM, CHEK2, PALB2, RAD51, XPD), have been implicated in some cases of breast cancer.


Assuntos
Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Feminino , Predisposição Genética para Doença , Recombinação Homóloga , Humanos , Mutação , Gravidez , Neoplasias de Mama Triplo Negativas/epidemiologia , Neoplasias de Mama Triplo Negativas/etiologia
3.
Cell Biochem Funct ; 37(7): 534-544, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418900

RESUMO

Chemotherapeutic drugs that induce DNA damage have the potential to kill cancer cells, but DNA repair protects cells from damage-induced cell death. Thus, eliminating DNA repair is a potential approach to overcome cell drug resistance. In this study, we observed that the gene expression of C-terminal binding protein interacting protein (CTIP) was promoted by TNF-α stimulation and prevented TNF-α-induced double-strand breaks (DSBs) in the genomes of cervical cancer cells. The putative miR-130b targeted site within 3' untranslated region (UTR) of CTIP mRNA was identified through in silico analysis and confirmed based on experimental data. By targeting the CTIP gene, miR-130b caused the accumulation of DSBs and accelerated cell apoptosis in combination with poly ADP ribose polymerase (PARP) inhibitors. Additionally, overexpression of the CTIP gene elevated cancer cell viability by promoting proliferation while miR-130b antagonized CTIP-stimulated cell reproduction. Consequently, miR-130b destruction of DNA repair should be employed as a strategy to treat cervical cancer. SIGNIFICANCE OF THE STUDY: Cervical cancer threatens the health of women all over the world. In this study, we observed that miR-130b was able to cause the accumulation of DNA double-strand breaks through suppressing the gene expression of C-terminal binding protein interacting protein and to accelerate cell apoptosis by preventing DNA damage repairs in cervical cancer cells. As far as we know, the impact of miR-130b on the DNA double-strand break repair and on the cell apoptosis induced by the destruction of DNA repair in cervical cancer cells was firstly documented. It is reasonable to believe that miR-130b destruction of DNA repair may be employed as a strategy to treat cervical cancer in the future.


Assuntos
Oxirredutases do Álcool/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias do Colo do Útero/metabolismo , Reparo do DNA , Feminino , Células HeLa , Humanos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/patologia
4.
DNA Cell Biol ; 38(10): 1030-1039, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368785

RESUMO

Both endogenous and exogenous factors can cause DNA damage that compromises genomic integrity and cell viability. A proper DNA damage response (DDR) plays a role in maintaining genome stability and preventing tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion, whose response is dominated by the ataxia-telangiectasia mutated (ATM) protein kinase. After being activated by the sensor Mre11-Rad50-Nbs1 (MRN) complex or acetyltransferase Tip60, ATM rapidly phosphorylates downstream targets to launch DDR signaling when DNA is damaged. However, the exact mechanism of DDR is complex and ambiguous. Ufmylation, one type of ubiquitin-like modification, proceeds mainly through a three-step enzymatic reaction to help ubiquitin-fold modifier 1 (Ufm1), attach to substrates with ubiquitin-like modifier-activating enzyme 5 (Uba5), Ufm1-conjugating enzyme 1 (Ufc1) and Ufm1-specific ligase 1 (Ufl1). Although ubiquitination is essential to the DSBs response, the potential function of ufmylation in DDR is largely unknown. Herein, we review the relationship between ufmylation and DDR to elucidate the function and mechanism of ufmylation in DDR, which would reveal the pathogenesis of some diseases and provide new guidance to create a therapeutic method.


Assuntos
Doenças Cardiovasculares/metabolismo , Quebras de DNA de Cadeia Dupla , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Esquizofrenia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Reparo do DNA , Genoma Humano , Instabilidade Genômica , Humanos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Proteínas/genética , Esquizofrenia/genética , Esquizofrenia/patologia , Transdução de Sinais , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
5.
Genes Dev ; 33(17-18): 1175-1190, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31395742

RESUMO

The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Ribossômico/genética , Regulação da Expressão Gênica/genética , RNA Longo não Codificante/metabolismo , Nucléolo Celular/metabolismo , Histonas/metabolismo , Recombinação Homóloga/genética , Membrana Nuclear/metabolismo
6.
Nat Commun ; 10(1): 2954, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273204

RESUMO

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cromatina/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Recombinação Homóloga/genética , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
7.
DNA Cell Biol ; 38(8): 747-753, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31188020

RESUMO

Aberrant neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis. While the genotoxic function of PMNs and its implications in carcinogenesis have been primarily associated with oxidative stress, recent work by Butin-Israeli and colleagues has defined a novel mechanism where PMN-derived microparticles through the delivery and activity of specific miRNAs promoted formation of double-strand breaks (DSBs), and in parallel, suppressed DSB repair through the downregulation of lamin B1 and Rad51. Respective downregulation of these two proteins compromised the nuclear envelope and high-fidelity repair by homologous recombination, increasing DSB accumulation and aneuploidy. This discovery defined a novel mode of action where PMN-mediated suppression of DSB repair leading to genomic instability in the injured mucosa may facilitate progression toward colorectal cancer.


Assuntos
Instabilidade Genômica , Doenças Inflamatórias Intestinais/tratamento farmacológico , Neutrófilos/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/prevenção & controle , Quebras de DNA de Cadeia Dupla , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , MicroRNAs/metabolismo , Neutrófilos/fisiologia , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
8.
Nat Commun ; 10(1): 2862, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253793

RESUMO

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.


Assuntos
Cromátides/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/genética , Troca de Cromátide Irmã , Telófase/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Nat Commun ; 10(1): 2866, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253785

RESUMO

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA , Edição de Genes/métodos , Reparo de DNA por Recombinação/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Mutagênese Insercional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
10.
Cancer Sci ; 110(8): 2471-2484, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187548

RESUMO

Endoplasmic reticulum stress (ERS) plays a key role in the pathogenesis and development of tumors and protects tumor cells from radiation damage and drug-induced stress. We previously demonstrated that EGFR confers radioresistance in human papillomavirus (HPV)-negative human oropharyngeal carcinoma by activating ERS signaling through PERK and IRE1α. In addition, PERK confers radioresistance by activating the inflammatory cytokine NF-κB. However, the effect of IRE1 on radiosensitivity has not yet been fully elucidated. Here, we clarified that IRE1 overexpression was associated with poor outcome in HPV-negative patients treated with radiotherapy (P = 0.0001). In addition, a significantly higher percentage of radioresistant HPV-negative patients than radiosensitive HPV-negative patients exhibited high IRE expression (66.7% vs 27.8%, respectively; P = 0.001). Silencing IRE1 and XBP1 increased DNA double-strand break (DSB) and radiation-induced apoptosis, thereby increasing the radiosensitivity of HPV-negative oropharyngeal carcinoma cells. IRE1-XBP1 silencing also inhibited radiation-induced IL-6 expression at both the RNA and protein levels. The regulatory effect of IRE1-XBP1 silencing on DNA DSB-induced and radiation-induced apoptosis was inhibited by pretreatment with IL-6. These data indicate that IRE1 regulates radioresistance in HPV-negative oropharyngeal carcinoma through IL-6 activation, enhancing X-ray-induced DNA DSB and cell apoptosis.


Assuntos
Endorribonucleases/metabolismo , Interleucina-6/metabolismo , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância a Radiação/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Apoptose/fisiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Humanos , NF-kappa B/metabolismo , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
11.
PLoS Genet ; 15(6): e1008177, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170160

RESUMO

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Prófase Meiótica I/genética , Proteínas MutL/genética , Animais , Pareamento Cromossômico/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Homóloga/genética , Masculino , Meiose/genética , Camundongos , Proteína 1 Homóloga a MutL/genética , Proteínas MutS/genética , Rad51 Recombinase/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo
12.
Environ Pollut ; 252(Pt A): 917-923, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31226516

RESUMO

Glyphosate-based herbicides (GBH) are the most widely used pesticides in the world. The extensive use of them increases the potential human health risk, including the human inhalation toxicity risk. We studied the effect of the most famous GBH Roundup® (RDP) in the concentration range from 50 to 125 µg/mL on Mitochondria-Associated apoptosis and DNA damage in Human alveolar carcinoma cells (A549 cells). Alkaline comet assay, immunofluorescence assay and Flow Cytometric Analysis assay were employed to detect DNA damages and apoptosis of A549 cells. We found RDP caused concentration-dependent increases in DNA damages and proportion of apoptotic cells in A549 cells. RDP induced the DNA single-strand breaks and double-strand breaks; the collapse of mitochondrial membrane by increasing Bax/Bcl-2, resulting in the release of cytochrome c into cytosol and then activated caspase-9/-3, cleaved poly (ADP-ribose) polymerase (PARP) in human lung tissue cells. The results demonstrate that RDP can induce A549 cells cytotoxic effects in vitro at the concentration lower than the occupational exposures level of workers, which means RDP has a potential threat to human health.


Assuntos
Apoptose/efeitos dos fármacos , Glicina/análogos & derivados , Herbicidas/toxicidade , Pulmão/patologia , Mitocôndrias/efeitos dos fármacos , Células A549 , Adenocarcinoma Bronquioloalveolar/genética , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Ensaio Cometa , Citocromos c/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Glicina/toxicidade , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
13.
J Cell Biol ; 218(7): 2075-2076, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31189608

RESUMO

The close interplay between DNA replication and repair is underscored by a report from Chen et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808134) in this issue. The authors demonstrate that the non-homologous end-joining factor XLF promotes the stability of replication forks.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA
14.
Nat Commun ; 10(1): 2535, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182712

RESUMO

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Acilação , Reparo do DNA , Membrana Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo
15.
Eur J Med Chem ; 175: 330-348, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096154

RESUMO

Human DNA topoisomerases represent one of the key targets of modern chemotherapy. An emerging group of catalytic inhibitors of human DNA topoisomerase IIα comprises a new paradigm directed to circumvent the known limitations of topoisomerase II poisons such as cardiotoxicity and induction of secondary tumors. In our previous studies, 4,6-substituted-1,3,5-triazin-2(1H)-ones were discovered as catalytic inhibitors of topo IIα. Here, we report the results of our efforts to optimize several properties of the initial chemical series that did not exhibit cytotoxicity on cancer cell lines. Using an optimized synthetic route, a focused chemical library was designed aimed at further functionalizing substituents at the position 4 of the 1,3,5-triazin-2(1H)-one scaffold to enable additional interactions with the topo IIα ATP binding site. After virtual screening, selected 36 analogues were synthesized and experimentally evaluated for human topo IIα inhibition. The optimized series displayed improved inhibition of topo IIα over the initial series and the catalytic mode of inhibition was confirmed for the selected active compounds. The optimized series also showed cytotoxicity against HepG2 and MCF-7 cell lines and did not induce double-strand breaks, thus displaying a mechanism of action that differs from the topo II poisons on the cellular level. The new series represents a new step in the development of the 4,6-substituted-1,3,5-triazin-2(1H)-one class towards novel efficient anticancer therapies utilizing the catalytic topo IIα inhibition paradigm.


Assuntos
DNA Topoisomerases Tipo II/efeitos dos fármacos , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Triazinas/química , Triazinas/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Catálise , Quebras de DNA de Cadeia Dupla , Células Hep G2 , Histonas/metabolismo , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/metabolismo
16.
Mol Biol (Mosk) ; 53(2): 311-323, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099781

RESUMO

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Vírus da Hepatite B , Sistemas CRISPR-Cas/genética , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/genética
17.
Nat Genet ; 51(6): 1011-1023, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110352

RESUMO

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.


Assuntos
Quebras de DNA de Cadeia Dupla , Loci Gênicos , Neoplasias/genética , Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Elementos Facilitadores Genéticos , Etoposídeo/farmacologia , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Íntrons , Neoplasias/patologia , Regiões Promotoras Genéticas , Sítios de Splice de RNA , Inibidores da Topoisomerase/farmacologia , Sítio de Iniciação de Transcrição
18.
Eur J Med Chem ; 176: 492-512, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31132480

RESUMO

We report the design, synthesis and biological studies on a group of mixed ligand Fe(III) complexes as anti-cancer drug candidates, namely their interaction with DNA, cytotoxicity and mechanism(s) of action. The aim is to obtain stable, efficient and selective Fe-complexes to be used as anti-cancer agents with less damaging side effects than previously reported compounds. Five ternary Fe(III) complexes bearing a tripodal aminophenolate ligand L2-, H2L = N,N-bis(2-hydroxy-3,5-dimethylbenzyl)-N-(2-pyridylmethyl)amine, and different aromatic bases NN = 2,2'-bipyridine [Fe(L)(bipy)]PF6 (1), 1,10-phenanthroline [Fe(L)(phen)]PF6 (2), or a phenanthroline derivative co-ligand: [Fe(L)(amphen)]NO3 (3), [Fe(L)(amphen)]PF6 (3a), [Fe(L)(Clphen)]PF6 (4), [Fe(L)(epoxyphen)]PF6 (5) (where amphen = 1,10-phenanthroline-5-amine, epoxyphen = 5,6-epoxy-5,6-dihydro-1,10-phenanthroline, Clphen = 5-chloro-1,10-phenanthroline) and the [Fe(L)(EtOH)]NO3 (6) complex are synthesized. The compounds are characterized in the solid state and in solution by elemental analysis, ESI-MS, magnetic susceptibility measurements and FTIR, UV-Vis, 1H and 13C NMR and fluorescence spectroscopies. [Fe(phen)Cl3] and [Fe(amphen)Cl3] were also prepared for comparison purposes. Spectroscopic binding studies indicate groove binding as the main interaction for most complexes with DNA, and for those containing amphen a B- to Z-DNA conformational change is proposed to occur. As determined via MTT analysis all compounds 1-6 are cytotoxic against a panel of three different cell lines (HeLa, H1299, MDA-MB-231). For selected compounds with promising cytotoxic activity, apoptosis was evaluated using cell and DNA morphology, TUNEL, Annexin V/7AAD staining and caspase3/7 activity. The compounds induce oxidative DNA damage on plasmid DNA and in cell culture as assessed by 8-oxo-Guanine and γH2AX staining. Comet assay confirmed the presence of genomic damage. There is also increased reactive oxygen species formation following drug treatment, which may be the relevant mechanism of action, thus differing from that normally assumed for cisplatin. The Fe(III)-complexes were also tested against strains of M. Tuberculosis (MTb), complex 2 depicting higher anti-MTb activity than several known second line drugs. Hence, these initial studies show prospective anti-cancer and anti-MTb activity granting promise for further studies.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Ferro/química , Fenantrolinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Antituberculosos/síntese química , Antituberculosos/química , Antituberculosos/farmacologia , Antituberculosos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , DNA/química , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Desenho de Drogas , Estabilidade de Medicamentos , Humanos , Ligantes , Mycobacterium tuberculosis/efeitos dos fármacos , Fenantrolinas/síntese química , Fenantrolinas/química , Fenantrolinas/toxicidade , Estudos Prospectivos , Espécies Reativas de Oxigênio/metabolismo
19.
Genes Dev ; 33(11-12): 684-704, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048545

RESUMO

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
20.
Radiat Res ; 192(1): 1-12, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31081741

RESUMO

We present a comprehensive comparative analysis on the repair of radiation-induced DNA damage ex vivo in 15 strains of mice, including 5 inbred reference strains and 10 collaborative-cross strains, of both sexes, totaling 5 million skin fibroblast cells imaged by three-dimensional highthroughput conventional microscopy. Non-immortalized primary skin fibroblasts derived from 76 mice were subjected to increasing doses of both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe), which are relevant to carcinogenesis and human space exploration. Automated image quantification of 53BP1 radiation-induced foci (RIF) formation and repair during the first 4-48 h postirradiation was performed as a function of dose and LET. Since multiple DNA double-strand breaks (DSBs) are induced in a dose- and LET-dependent manner, our data suggest that when DSBs are formed within the same discrete nuclear region, referred to as the "repair domain", novel mathematical formalisms used to report RIF allowed us to conclude that multiple DSBs can be present in single RIF. Specifically, we observed that the number of RIF per Gy was lower for higher X-ray doses or higher LET particles (i.e., 600 MeV/n 56Fe), suggesting there are more DSBs per RIF when the local absorbed dose increases in the nucleus. The data also clearly show that with more DSBs per RIF, it becomes more difficult for cells to fully resolve RIF. All 15 strains showed the same dose and LET dependence, but strain differences were preserved under various experimental conditions, indicating that the number and sizes of repair domains are modulated by the genetic background of each strain.


Assuntos
Dano ao DNA , Transferência Linear de Energia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Cinética , Camundongos , Especificidade da Espécie , Fatores de Tempo , Raios X/efeitos adversos
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