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1.
Arch Insect Biochem Physiol ; 102(1): e21598, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31290186

RESUMO

At present, the effect of ultraviolet (UV) radiation on the interaction between Bombyx mori nucleopolyhedrovirus (BmNPV) and host remains unclear. In the current study, UV treatment significantly reduced the activity of BmNPV budded viruses (BVs), and UV-damaged BmN cells were not conducive to BmNPV proliferation. BmNPV infection significantly reduced the viability of host cells, but increased the viability of high-dose UV-treated host cells. Furthermore, the quantitative reverse-transcription PCR (qPCR) results suggested that BmNPV and Bombyx mori might mutually use the same DNA repair proteins for repairing UV-induced damage and BmNPV infection promote the ability of host cells to repair UV-induced damage.


Assuntos
Bombyx/virologia , Interações Hospedeiro-Patógeno/efeitos da radiação , Nucleopolyhedrovirus/efeitos da radiação , Animais , Bombyx/imunologia , Bombyx/metabolismo , Bombyx/efeitos da radiação , Sobrevivência Celular , Células Cultivadas , Endonucleases Flap/metabolismo , Neuropeptídeos/metabolismo , Raios Ultravioleta
2.
Nat Commun ; 10(1): 2420, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160570

RESUMO

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Endonucleases Flap/metabolismo , Humanos , Hidrólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Nat Commun ; 10(1): 2104, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068591

RESUMO

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.


Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
4.
Nanoscale ; 11(8): 3633-3638, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30741288

RESUMO

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 µL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.


Assuntos
DNA Bacteriano/análise , Endonucleases Flap/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Chlamydia trachomatis/genética , DNA/análise , DNA/sangue , DNA/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/sangue , DNA de Cadeia Simples/metabolismo , Humanos , Limite de Detecção , Ácidos Nucleicos/sangue , Ácidos Nucleicos/metabolismo , Compostos Orgânicos/química
5.
J Agric Food Chem ; 67(6): 1656-1665, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30694659

RESUMO

Human flap endonuclease 1 (hFEN1) is instrumental in DNA replication and repair. It is able to cleave the 5' single-stranded protrusion (also known as 5' flap) resulting from strand displacement reactions. In light of its crucial functions, hFEN1 is now deemed as a nontrivial target in the DNA damage response system for anticancer drug development. Herein, we report that myricetin and some natural flavonoids are able to inhibit hFEN1. Structure-activity relationship, inhibitory mechanisms, molecular docking, and cancer cell-based assays have been performed. Our original findings expand the activity of flavonoids and may pave the way for flavonoid-assisted targeted cancer therapy.


Assuntos
Neoplasias do Colo/enzimologia , Inibidores Enzimáticos/química , Endonucleases Flap/antagonistas & inibidores , Flavonoides/química , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Inibidores Enzimáticos/farmacologia , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Flavonoides/farmacologia , Células HT29 , Humanos , Simulação de Acoplamento Molecular
6.
Mol Cell ; 73(5): 885-899.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30686591

RESUMO

BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.


Assuntos
Proteína BRCA2/genética , Sistemas CRISPR-Cas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases Flap/genética , Genes Letais , Neoplasias/genética , Interferência de RNA , Mutações Sintéticas Letais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética
7.
Am J Pathol ; 188(1): 242-251, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29037854

RESUMO

Flap endonuclease 1 (FEN1) plays a crucial role in both DNA replication and damage repair. In this study, FEN1 expression and its clinical-pathologic significance in non-small-cell lung cancer (NSCLC) was investigated. Quantitative RT-PCR and immunohistochemistry analysis identified that both FEN1 mRNA and protein were highly overexpressed in about 36% of 136 cancer tissues compared to adjacent tissues, in which FEN1 was generally undetectable. Notably, patients with FEN1-overexpressed cancers were prone to have poor differentiation and poor prognosis. A strong positive correlation between the levels of FEN1 and Ki-67 staining was identified in these NSCLC tissues (r = 0.485), suggesting overexpressed FEN1 conferred a proliferative advantage to NSCLC. Furthermore, knockdown of FEN1 resulted in G1/S or G2/M phase cell cycle arrest and suppressed in vitro cellular proliferation in NSCLC cancer cells. Consistently, a selective FEN1 inhibitor was shown to effectively inhibit cellular proliferation of NSCLC cells in a dose-dependent manner. Additionally, knockdown of FEN1 significantly attenuated homologous DNA repair efficiency and enhanced cytotoxic effects of cisplatin in NSCLC cells. Taken together, these findings have indicated that overexpressed FEN1 represents a prognostic biomarker and potential therapeutic target for NSCLC treatment, which warrants further study.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/genética , Endonucleases Flap/metabolismo , Neoplasias Pulmonares/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Feminino , Endonucleases Flap/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Regulação para Cima
8.
BMC Biol ; 15(1): 90, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28969641

RESUMO

BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Recombinação Genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Semin Immunol ; 33: 3-15, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29042025

RESUMO

Leukotriene B4 (LTB4) is a lipid mediator derived from arachidonic acid (AA) by the sequential action of 5-lipoxygenase (5-LOX), 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase (LTA4H). It was initially recognized for its involvement in the recruitment of neutrophils and is one of the most potent chemotactic agents known to date. A large body of data has indicated that LTB4 plays a significant role in many chronic inflammatory diseases, such as arthritis, chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer and more recently, metabolic disorder. In this review, we focus on the biosynthesis of LTB4 and its biological effects. In particular, we will describe a basic biochemical understanding integrated with recent developments in the field of structural biology of the three key enzymes (5-LOX, FLAP and LTA4H) in LTB4 biosynthesis, and also summarize the most outstanding work on in vivo biological and pathogenic roles of these enzymes and the development of enzyme inhibitors.


Assuntos
Artrite/imunologia , Doenças Cardiovasculares/imunologia , Leucotrieno B4/biossíntese , Neoplasias/imunologia , Neutrófilos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Endonucleases Flap/metabolismo , Humanos , Relação Estrutura-Atividade
10.
Cell Rep ; 20(7): 1553-1562, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28813668

RESUMO

Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structure-specific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4. Recruitment takes place into a class of stress foci defined by Cmr1/WDR76, a protein involved in preserving genome stability, and depends on the E2-ubiquitin-conjugating enzyme Rad6 and the E3-ubiquitin ligase Bre1. Foci dynamics show that, in the presence of DNA intermediates that need resolution by Mus81-Mms4, Mus81 foci persist until this endonuclease is activated by Mms4 phosphorylation. Our data suggest that subnuclear relocalization is relevant for the function of Mus81-Mms4 and, probably, of the endonucleases that colocalize with it.


Assuntos
Reparo do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
11.
DNA Repair (Amst) ; 57: 66-75, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28704715

RESUMO

Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are critical to preserve genome integrity. In addition to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Endonucleases Flap/genética , Instabilidade Genômica , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/genética , Linhagem Celular , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima
12.
PLoS One ; 12(7): e0180153, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28678842

RESUMO

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/fisiologia , Endonucleases Flap/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Acetiltransferases/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Dano ao DNA , DNA Fúngico/genética , DNA Mitocondrial/genética , Ativação Enzimática , Endonucleases Flap/metabolismo , Técnicas de Inativação de Genes , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Mutagênese , Mutação Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Mol Biosyst ; 13(8): 1630-1639, 2017 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-28685785

RESUMO

Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Aprendizado de Máquina , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/enzimologia , Colo/patologia , Bases de Dados de Compostos Químicos , Descoberta de Drogas , Inibidores Enzimáticos/química , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Concentração Inibidora 50 , Naftoquinonas/química , Naftoquinonas/farmacologia , Especificidade de Órgãos
14.
PLoS One ; 12(6): e0179278, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28628639

RESUMO

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.


Assuntos
Reparo do DNA/efeitos dos fármacos , Endonucleases Flap/metabolismo , Hidroxiureia/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/toxicidade , DNA/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Humanos , Hidroxiureia/química , Proteína Homóloga a MRE11 , Instabilidade de Microssatélites/efeitos dos fármacos , Mutação , Ftalazinas/toxicidade , Piperazinas/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases/toxicidade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética
15.
Mol Oncol ; 11(6): 640-654, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28371273

RESUMO

Lung cancer is one of the leading causes of cancer mortality worldwide. The therapeutic effect of chemotherapy is limited due to the resistance of cancer cells, which remains a challenge in cancer therapeutics. In this work, we found that flap endonuclease 1 (FEN1) is overexpressed in lung cancer cells. FEN1 is a major component of the base excision repair pathway for DNA repair systems and plays important roles in maintaining genomic stability through DNA replication and repair. We showed that FEN1 is critical for the rapid proliferation of lung cancer cells. Suppression of FEN1 resulted in decreased DNA replication and accumulation of DNA damage, which subsequently induced apoptosis. Manipulating the amount of FEN1 altered the response of lung cancer cells to chemotherapeutic drugs. A small-molecule inhibitor (C20) was used to target FEN1 and this enhanced the therapeutic effect of cisplatin. The FEN1 inhibitor significantly suppressed cell proliferation and induced DNA damage in lung cancer cells. In mouse models, the FEN1 inhibitor sensitized lung cancer cells to a DNA damage-inducing agent and efficiently suppressed cancer progression in combination with cisplatin treatment. Our study suggests that targeting FEN1 may be a novel and efficient strategy for a tumor-targeting therapy for lung cancer.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Endonucleases Flap/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células , Cisplatino/farmacologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , Progressão da Doença , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto
16.
J Bacteriol ; 199(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28416706

RESUMO

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase.IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.


Assuntos
Exorribonucleases/metabolismo , Endonucleases Flap/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Thermococcus/enzimologia , Exorribonucleases/genética , Endonucleases Flap/genética , Deleção de Genes , Genoma Bacteriano , Viabilidade Microbiana/genética , Thermococcus/genética , Thermococcus/metabolismo
17.
J Biol Chem ; 292(12): 4777-4788, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28159842

RESUMO

During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.


Assuntos
Primers do DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases Flap/metabolismo , RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Transdução de Sinais , DNA/análise , DNA/genética , DNA/ultraestrutura , Primers do DNA/análise , Primers do DNA/genética , Replicação do DNA , DNA Fúngico/análise , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/análise , Endodesoxirribonucleases/genética , Endonucleases Flap/análise , Endonucleases Flap/genética , Mutação , RNA/análise , RNA/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/genética
18.
Elife ; 62017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230529

RESUMO

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.


Assuntos
DNA/metabolismo , Endonucleases Flap/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula , Especificidade por Substrato
19.
Oncotarget ; 8(16): 27593-27602, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28187440

RESUMO

Protein-protein interaction (PPI) plays a key role in cellular communication, Protein-protein interaction connected with each other with hubs and nods involved in signaling pathways. These interactions used to develop network based biomarkers for early diagnosis of cancer. FEN1(Flap endonuclease 1) is a central component in cellular metabolism, over expression and decrease of FEN1 levels may cause cancer, these regulation changes of Flap endonuclease 1reported in many cancer cells, to consider this data may needs to develop a network based biomarker. The current review focused on types of PPI, based on nature, detection methods and its role in cancer. Interacting partners of Flap endonuclease 1 role in DNA replication repair and development of anticancer therapeutics based on Protein-protein interaction data.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Descoberta de Drogas , Endonucleases Flap/metabolismo , Animais , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
20.
EMBO J ; 36(5): 664-678, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28096179

RESUMO

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Ativação Enzimática , Saccharomyces cerevisiae/enzimologia
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