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1.
Nat Commun ; 11(1): 5746, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184279

RESUMO

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Culina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Mitose/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968016

RESUMO

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA , Diploide , Feminino , Endonucleases Flap/genética , Genoma Fúngico/genética , Humanos , Perda de Heterozigosidade/genética , Proteínas de Membrana/genética , Peroxirredoxinas/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
3.
PLoS Genet ; 16(7): e1008933, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692737

RESUMO

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases Flap/genética , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Fase S/genética , Schizosaccharomyces/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
4.
Proc Natl Acad Sci U S A ; 117(32): 19415-19424, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719125

RESUMO

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.


Assuntos
Antineoplásicos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/genética , Instabilidade Genômica/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Life Sci ; 257: 118089, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659369

RESUMO

AIM: Hepatitis B virus (HBV) is a major cause of a variety of liver diseases. Existing antiviral drugs cannot eradicate HBV from our body, and the main reason is unclear on the molecular mechanism of HBV replication. Flap endonuclease 1 (FEN1) can repair relaxed circular DNA (HBV rcDNA) to covalently closed circular DNA (HBV cccDNA) that promotes HBV DNA replication, while its specific regulatory detail remains unclear. In addition, miR-146a is close related to regulation in HBV replication. This study aims to explore whether miR-146a regulates HBV cccDNA formation through FEN1. MAIN METHODS: We investigated the expression of miR-146a, FEN1 and HBV copies in HBV stable replication cell line HepG2.2.15 and its parent cell line HepG2 transfected miR-146a and FEN1 plasmid by qRT-PCR and western blot, to identify the cooperation of Argonaute-2 (Ago2) and miR-146a by Ago2 siRNA and Ago2 RNA Binding Protein Immunoprecipitation (RIP). KEY FINDINGS: Compared with the control group, we found that the expression of miR-146a was significantly up-regulated in HepG2.2.15, and the expression of FEN1 and HBV copies were also significantly up-regulated. On contrary, the expression of target gene of miR-146a, interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor-6 (TRAF6), was significantly decreased in HepG2.2.15. With the use of Ago2 siRNA and then Ago2 RIP, we found that Ago2 performed as a carrier for miR-146a to promote HBV replication. SIGNIFICANCE: The results suggest a novel miR-146a â†’ FEN1 â†’ HBV DNA regulatory axis in HBV replication life. Ago2 cooperates with miR-146a to regulate the transcription and expression level of FEN1 protein through the downstream target gene IRAK1/TRAF6, and to promote HBV replication.


Assuntos
Proteínas Argonauta/genética , Vírus da Hepatite B/fisiologia , MicroRNAs/genética , Replicação Viral/genética , DNA Circular/genética , DNA Viral/genética , Endonucleases Flap/genética , Células Hep G2 , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética
6.
PLoS Genet ; 16(5): e1008816, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469862

RESUMO

Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Telomerase/deficiência , Replicação do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Viabilidade Microbiana , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telômero/metabolismo , Homeostase do Telômero
7.
Cancer Res ; 80(10): 1914-1926, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32193286

RESUMO

Estrogen receptor α (ERα) is a key transcriptional regulator in the majority of breast cancers. ERα-positive patients are frequently treated with tamoxifen, but resistance is common. In this study, we refined a previously identified 111-gene outcome prediction-classifier, revealing FEN1 as the strongest determining factor in ERα-positive patient prognostication. FEN1 levels were predictive of outcome in tamoxifen-treated patients, and FEN1 played a causal role in ERα-driven cell growth. FEN1 impacted the transcriptional activity of ERα by facilitating coactivator recruitment to the ERα transcriptional complex. FEN1 blockade induced proteasome-mediated degradation of activated ERα, resulting in loss of ERα-driven gene expression and eradicated tumor cell proliferation. Finally, a high-throughput 465,195 compound screen identified a novel FEN1 inhibitor, which effectively blocked ERα function and inhibited proliferation of tamoxifen-resistant cell lines as well as ex vivo-cultured ERα-positive breast tumors. Collectively, these results provide therapeutic proof of principle for FEN1 blockade in tamoxifen-resistant breast cancer. SIGNIFICANCE: These findings show that pharmacologic inhibition of FEN1, which is predictive of outcome in tamoxifen-treated patients, effectively blocks ERα function and inhibits proliferation of tamoxifen-resistant tumor cells.


Assuntos
Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/metabolismo , Endonucleases Flap/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Endonucleases Flap/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Tamoxifeno/uso terapêutico
8.
Nucleic Acids Res ; 48(6): 3165-3180, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32034423

RESUMO

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain-especially the O helix-to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO-POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.


Assuntos
DNA Polimerase I/genética , DNA Polimerase Dirigida por DNA/genética , Endonucleases Flap/genética , Fosfodiesterase I/genética , Sítios de Ligação , Cristalografia por Raios X , DNA Polimerase I/química , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/química , Endonucleases Flap/química , Magnésio/química , Mycobacterium/enzimologia , Mycobacterium/genética , Conformação de Ácido Nucleico , Nucleotídeos/genética , Fosfodiesterase I/química
9.
Folia Microbiol (Praha) ; 65(2): 407-415, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31401764

RESUMO

Flap endonuclease is a structure-specific nuclease which cleaves 5'-flap of bifurcated DNA substrates. Genome sequence of Thermococcus kodakarensis harbors an open reading frame, Tk1281, exhibiting high homology with archaeal flap endonucleases 1. The corresponding gene was cloned and expressed in Escherichia coli, and the gene product was purified to apparent homogeneity. Tk1281 was a monomer of 38 kDa and catalyzed the cleavage of 5'-flap from double-stranded DNA substrate containing single-stranded DNA flap. The highest cleavage activity was observed at 80 °C and pH 7.5. Under optimal conditions, Tk1281 exhibited apparent Vmax and Km values of 278 nmol/min/mg and 37 µM, respectively, against a 54-nucleotide double-stranded substrate containing a single-stranded 5'-flap of 27 nucleotides. A unique feature of Tk1281 is its highest activation in the presence of Co2+ and no activation with Mn2+. To the best of our knowledge, this is the first cloning and characterization of a flap endonuclease from the genus Thermococcus.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular , Endonucleases Flap/genética , Thermococcus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Cinética , Peso Molecular , Especificidade por Substrato , Thermococcus/química , Thermococcus/genética
10.
Aging Cell ; 19(2): e13084, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31854076

RESUMO

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age-dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high-throughput replica-pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age-specific mutation suppression gene. While wild-type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Senescência Celular/genética , Instabilidade Genômica/genética , Proteínas de Membrana/genética , Taxa de Mutação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Replicação do DNA/genética , Endonucleases Flap/genética , Ontologia Genética , Técnicas Genéticas , Mutagênese , Mutação , Acúmulo de Mutações , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Saccharomyces cerevisiae/fisiologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
11.
Oncogene ; 39(1): 234-247, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31471584

RESUMO

An increased DNA repair capacity is associated with drug resistance and limits the efficacy of chemotherapy in breast cancers. Flap endonuclease 1 (FEN1) participates in various DNA repair pathways and contributes to cancer progression and drug resistance in chemotherapy. Inhibition of FEN1 serves as a potent strategy for cancer therapy. Here, we demonstrate that microRNA-140 (miR-140) inhibits FEN1 expression via directly binding to its 3' untranslated region, leading to impaired DNA repair and repressed breast cancer progression. Overexpression of miR-140 sensitizes breast cancer cells to chemotherapeutic agents and overcomes drug resistance in breast cancer. Notably, ectopic expression of FEN1 abates the effects of miR-140 on DNA damage and the chemotherapy response in breast cancer cells. Furthermore, the transcription factor/repressor Ying Yang 1 (YY1) directly binds to the miR-140 promoter and activates miR-140 expression, which is attenuated in doxorubicin resistance. Our results demonstrate that miR-140 acts as a tumor suppressor in breast cancer by inhibiting FEN1 to repress DNA damage repair and reveal miR-140 to be a new anti-tumorigenesis factor for adjunctive breast cancer therapy. This novel mechanism will enhance the treatment effect of chemotherapy in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Endonucleases Flap/genética , MicroRNAs/genética , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Regiões Promotoras Genéticas/genética
12.
Cancer Med ; 8(18): 7774-7780, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31670906

RESUMO

BACKGROUND: Cervical cancer is one of the most common causes of cancer-associated mortality among affected women in the world. At present, treatment with weekly cisplatin plus ionizing radiation (IR) therapy is the standard regimen for cervical cancer, especially for locally advanced cervical cancer. The purpose of this study is to determine whether FEN1 inhibitors could enhance the therapeutic effect of IR therapy. METHODS: Western blot was applied to determine the expression of FEN1- and apoptosis-related proteins. Cell growth inhibition assay and colony formation assay were used to determine the effects of FEN1 inhibitor and IR exposure for Hela cells in vitro. CRISPR technology was used to knockdown FEN1 expression level of 293T cells, and tumor xenograft in nude mice was employed to determine the effects of FEN1 inhibitor and IR exposure on tumor growth in vivo. RESULTS: Our data revealed that FEN1 is overexpressed in HeLa cell and can be upregulated further by IR. We also demonstrated that FEN1 inhibitor enhances IR sensitivity of cervical cancer in vitro and in vivo. CONCLUSION: FEN1 inhibitor SC13 could sensitize radiotherapy of cervical cancer cell.


Assuntos
Inibidores Enzimáticos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Modelos Animais de Doenças , Feminino , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Camundongos , Radiação Ionizante , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Aging (Albany NY) ; 11(15): 5593-5612, 2019 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-31402791

RESUMO

Flap Endonuclease 1 (FEN1) is a known oncogene in an array of cancers, but its role in hepatocellular carcinoma (HCC) remains obscure. In this study, we report that FEN1 expression was elevated in the Cancer Genome Atlas (TCGA) database which was verified in HCC tissue and hepatoma cell lines. Pearson correlation analysis indicated that FEN1 was involved in HCC metastasis. We demonstrated that FEN1 silencing inhibits HCC cell epithelial-mesenchymal transition (EMT), invasion and migration in vitro and significantly suppressed tumor growth and metastasis in vivo. Conversely, FEN1 overexpression in HCC cells enhanced these metastatic processes. We further confirmed that FEN1 was a direct target of miR-140-5p, which was down-regulated in HCC tissues, and negatively correlated with FEN1 expression. Moreover, low miR-140-5p levels and high FEN1 expression predicted a poor clinical outcome. The effects of FEN1 overexpression could be partially abolished by miR-140-5p. miR-140-5p down-regulation and FEN1 overexpression were observed in a TGFß1 induced EMT model. TGFß1 mediated EMT could be blocked by miR-140-5p overexpression or FEN1 silencing. Taken together, our findings suggest that FEN1 is regulated by the TGFß1- miR-140-5p axis and promotes EMT in HCC.


Assuntos
Carcinoma Hepatocelular/genética , Endonucleases Flap/biossíntese , Neoplasias Hepáticas/genética , MicroRNAs/genética , Fator de Crescimento Transformador beta1/genética , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Endonucleases Flap/genética , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/genética , Transplante de Neoplasias , Prognóstico , Resultado do Tratamento , Regulação para Cima/genética
14.
FASEB J ; 33(10): 10717-10730, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31266372

RESUMO

Flap endonuclease 1 (FEN1) is recognized as a pivotal factor in DNA replication, long-patch excision repair, and telomere maintenance. Excessive FEN1 expression has been reported to be closely associated with cancer progression, but the specific mechanism has not yet been explored. In the present study, we demonstrated that FEN1 promoted breast cancer cell proliferation via an epigenetic mechanism of FEN1-mediated up-regulation of DNA methyltransferase (DNMT)1 and DNMT3a. FEN1 was proved to interact with DNMT3a through proliferating cell nuclear antigen (PCNA) to suppress microRNA (miR)-200a-5p expression mediated by methylation. Furthermore, miR-200a-5p was identified to repress breast cancer cell proliferation by inhibiting the expression of its target genes, hepatocyte growth factor (MET), and epidermal growth factor receptor (EGFR). Overall, our data surprisingly demonstrate that FEN1 promotes breast cancer cell growth via the formation of FEN1/PCNA/DNMT3a complex to inhibit miR-200a expression by DNMT-mediated methylation and to recover the target genes expression of miR-200a, MET, and EGFR. The novel epigenetic mechanism of FEN1 on proliferation promotion provides a significant clue that FEN1 might serve as a predictive biomarker and therapeutic target for breast cancer.-Zeng, X., Qu, X., Zhao, C., Xu, L., Hou, K., Liu, Y., Zhang, N., Feng, J., Shi, S., Zhang, L., Xiao, J., Guo, Z., Teng, Y., Che, X. FEN1 mediates miR-200a methylation and promotes breast cancer cell growth via MET and EGFR signaling.


Assuntos
Neoplasias da Mama/metabolismo , Endonucleases Flap/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética , Receptores ErbB/metabolismo , Feminino , Endonucleases Flap/antagonistas & inibidores , Endonucleases Flap/genética , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Células MCF-7 , Masculino , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Mutação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Mapas de Interação de Proteínas , Transdução de Sinais
15.
Comb Chem High Throughput Screen ; 22(6): 379-386, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31272350

RESUMO

AIM AND OBJECTIVE: Flap endonuclease-1 (FEN1) plays a central role in DNA replication and DNA damage repair process. In mammals, FEN1 functional sites variation is related to cancer and chronic inflammation, and supports the role of FEN1 as a tumor suppressor. However, FEN1 is overexpressed in multiple types of cancer cells and is associated with drug resistance, supporting its role as an oncogene. Hence, it is vital to explore the multi-functions of FEN1 in normal cell metabolic process. This study was undertaken to examine how the gene expression profile changes when FEN1 is downregulated in 293T cells. MATERIALS AND METHODS: Using the RNA sequencing and real-time PCR approaches, the transcript expression profile of FEN1 knockdown HEK293T cells have been detected for the next step evaluation, analyzation, and validation. RESULTS: Our results confirmed that FEN1 is important for cell viability. We showed that when FEN1 downregulation led to the interruption of nucleic acids related metabolisms, cell cycle related metabolisms are significantly interrupted. FEN1 may also participate in non-coding RNA processing, ribosome RNA processing, transfer RNA processing, ribosome biogenesis, virus infection and cell morphogenesis. CONCLUSION: These findings provide insight into how FEN1 nuclease might regulate a wide variety of biological processes, and laid the foundation for understanding the role of other RAD2 family nucleases in cell growth and metabolism.


Assuntos
Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Neoplasias/genética , Ácidos Nucleicos/metabolismo , Viroses/genética , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Endonucleases Flap/deficiência , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , RNA-Seq , Viroses/metabolismo , Viroses/patologia
16.
J Cell Biochem ; 120(8): 13583-13597, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30937972

RESUMO

Flap endonuclease 1 (FEN1) has emerged as an important enzyme in the maintenance of genomic instability and preventing carcinogenesis. The relationship between FEN1 -69G>A (rs174538)+4150G>T (rs4246215) polymorphisms and cancer susceptibility has been reported; however, results were inconclusive. In the present study, a meta-analysis of data from eligible reports was carried out to summarize the possible relationship between FEN1 polymorphisms and cancer risk. A total of 11 articles, including 20 studies with 7366 cases and 9028 controls and 18 studies with 6649 cases and 8325 controls for FEN1 rs174538 and FEN1 rs4246215 polymorphisms, respectively, were recruited for meta-analysis. Overall, meta-analyses showed that FEN1 rs174538 and rs4246215 polymorphisms are significantly associated with the decreased risk of cancer. The stratified analysis proposed that both variants were associated with protection against gastrointestinal cancer, breast cancer, hepatocellular cancer, esophageal cancer, gastric cancer, colorectal cancer, and lung cancer. In conclusion, this meta-analysis revealed an association between FEN1 polymorphisms and cancer risk. Additional studies in a larger study population that include subjects from a variety of ethnicities are warranted to further verify our findings.


Assuntos
Endonucleases Flap/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Neoplasias/genética , Genótipo , Haplótipos/genética , Humanos , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
17.
Clin Transl Oncol ; 21(8): 1026-1033, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30712236

RESUMO

PURPOSE: Flap endonuclease 1 (FEN1) is up-regulated by estrogen (17ß-estradiol, E2) and related to cisplatin resistance of human breast cancer cells. Letrozole, an aromatase inhibitor, suppresses the change of testosterone into estrogen and is frequently used to treat breast cancer. However, the effects of letrozole on FEN1 expression and cisplatin sensitivity in breast cancer cells overexpressing aromatase have not been revealed. METHODS: The expression of FEN1 and the proteins in ERK/Elk-1 signaling were evaluated by RT-PCR and Western blot. Cisplatin sensitivity was explored through CCK-8 and flow cytometry analysis, respectively. FEN1 siRNAs and FEN1 expression plasmid were transfected into cells to down-regulate or up-regulate FEN1 expression. The promotor activity of FEN1 was detected using luciferase reporter assay. RESULTS: FEN1 down-regulation improved cisplatin sensitivity of breast cancer cells overexpressing aromatase. Letrozole down-regulated FEN1 expression and increased cisplatin sensitivity. The sensitizing effect of letrozole to cisplatin was dependent on FEN1 down-regulation. FEN1 overexpression could block the sensitizing effect of letrozole to cisplatin. Testosterone up-regulated the promotor activity, protein expression of FEN1, and phosphorylation of ERK/Elk-1, which could be eliminated by both letrozole and MEK1/2 inhibitor U0126. Letrozole down-regulated FEN1 expression in an ERK/Elk-1-dependent manner. CONCLUSIONS: Our findings clearly demonstrate that letrozole improves cisplatin sensitivity of breast cancer cells overexpressing aromatase via down-regulation of FEN1 and suggest that a combined use of letrozole and cisplatin may be a potential treatment protocol for relieving cisplatin resistance in human breast cancer.


Assuntos
Aromatase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endonucleases Flap/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Letrozol/farmacologia , Antineoplásicos/farmacologia , Aromatase/química , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Regulação para Baixo , Feminino , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Humanos , Transdução de Sinais , Células Tumorais Cultivadas
18.
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 , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Enzimas Multifuncionais , 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
19.
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
20.
Nucleic Acids Res ; 47(4): 1814-1822, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30541106

RESUMO

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5' flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5' flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Exodesoxirribonucleases/genética , Endonucleases Flap/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , DNA/genética , Células Eucarióticas , Genoma Fúngico/genética , Saccharomyces cerevisiae/genética
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