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1.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(7): 936-941, 2020 Jul 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895148

RESUMO

OBJECTIVE: To observe the expression of HELQ and RAD51C in normal endometrial and endometrial stromal sarcoma (ESS) and analyze their correlation with the clinical features of the patients. METHODS: The expressions of HELQ and RAD51C proteins were detected by immunohistochemical staining in normal endometrial tissues (14 cases) and tumor tissues from patients with ESS (37 cases) treated in Hunan Provincial Cancer Hospital from January, 2013 to December, 2016. The correlations of the expressions of the two proteins with the patients'age, FIGO staging, tissue type, tumor size, and lymph node metastasis were analyzed. RESULTS: Immunohistochemical staining showed that the expressions of HELQ and RAD51C were both decreased in ESS patients compared with the normal group, and there was a positive correlation between HELQ and RAD51C expression (P < 0.05). HELQ expression in ESS was correlated with the tumor size and type. The expressions of HELQ and RAD51C were not correlated with the patients' age, FIGO stage and status of lymph node metastasis (P > 0.05). CONCLUSIONS: Homologous recombination- directed DNA repair involving HELQ and RAD51C may participate in the occurrence and progression of ESS.


Assuntos
Neoplasias do Endométrio , Sarcoma do Estroma Endometrial , DNA Helicases , Proteínas de Ligação a DNA , Endométrio , Feminino , Humanos , Metástase Linfática
2.
Nat Commun ; 11(1): 3419, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647123

RESUMO

The development and function of the brain require tight control of gene expression. Genome architecture is thought to play a critical regulatory role in gene expression, but the mechanisms governing genome architecture in the brain in vivo remain poorly understood. Here, we report that conditional knockout of the chromatin remodeling enzyme Chd4 in granule neurons of the mouse cerebellum increases accessibility of gene regulatory sites genome-wide in vivo. Conditional knockout of Chd4 promotes recruitment of the architectural protein complex cohesin preferentially to gene enhancers in granule neurons in vivo. Importantly, in vivo profiling of genome architecture reveals that conditional knockout of Chd4 strengthens interactions among developmentally repressed contact domains as well as genomic loops in a manner that tightly correlates with increased accessibility, enhancer activity, and cohesin occupancy at these sites. Collectively, our findings define a role for chromatin remodeling in the control of genome architecture organization in the mammalian brain.


Assuntos
Encéfalo/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Genoma , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/metabolismo , DNA Helicases/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Camundongos Knockout , Modelos Genéticos , Ligação Proteica
3.
Nat Commun ; 11(1): 3713, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709841

RESUMO

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA/química , DNA/metabolismo , Animais , Bacteriófago T4 , Microscopia Crioeletrônica , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Escherichia coli/genética
4.
PLoS One ; 15(7): e0235705, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32649682

RESUMO

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Interferons/farmacologia , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo
5.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 936-941, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701227

RESUMO

OBJECTIVE: To observe the expression of HELQ and RAD51C in normal endometrial and endometrial stromal sarcoma (ESS) and analyze their correlation with the clinical features of the patients. METHODS: The expressions of HELQ and RAD51C proteins were detected by immunohistochemical staining in normal endometrial tissues (14 cases) and tumor tissues from patients with ESS (37 cases) treated in Hunan Provincial Cancer Hospital from January, 2013 to December, 2016. The correlations of the expressions of the two proteins with the patients'age, FIGO staging, tissue type, tumor size, and lymph node metastasis were analyzed. RESULTS: Immunohistochemical staining showed that the expressions of HELQ and RAD51C were both decreased in ESS patients compared with the normal group, and there was a positive correlation between HELQ and RAD51C expression (P < 0.05). HELQ expression in ESS was correlated with the tumor size and type. The expressions of HELQ and RAD51C were not correlated with the patients' age, FIGO stage and status of lymph node metastasis (P > 0.05). CONCLUSIONS: Homologous recombination- directed DNA repair involving HELQ and RAD51C may participate in the occurrence and progression of ESS.


Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Neoplasias do Endométrio , Regulação Neoplásica da Expressão Gênica , Sarcoma do Estroma Endometrial , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/fisiopatologia , Endométrio/fisiopatologia , Feminino , Humanos , Metástase Linfática/fisiopatologia , Sarcoma do Estroma Endometrial/fisiopatologia
6.
PLoS One ; 15(6): e0234246, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32502225

RESUMO

INTRODUCTION: Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development. METHODS: To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal. RESULTS: In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants. CONCLUSION: Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.


Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Atresia Esofágica/genética , Exoma/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas Repressoras/genética , Fístula Traqueoesofágica/genética , Animais , Humanos , Camundongos , Sequenciamento Completo do Exoma
7.
Life Sci ; 256: 117820, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512012

RESUMO

Neuroblastoma (NB) is one of the most common malignant tumors in children. Chemotherapy resistance is one of the significant challenges in the treatment of high-risk NB patients, and it is necessary to search for new valid targets for NB treatment. This study aims to explore the possible role of PIF1 in NB by using bioinformatic analysis and downregulation of PIF1 with specific siRNA. Kyoto genome encyclopedia and R language based gene ontology was used to analyze the differentially expressed genes (DEGs) (including PIF1) when MYCN expression was silenced in NB cells. Analysis based on the R2 database showed a lower expression of PIF1 correlated with good prognosis in NB patients. Downregulation of MYCN expression by transfecting MYCN siRNA (#1, #2) into NB cells decreased the PIF1 expression at both mRNA and protein levels, while upregulation of MYCN expression by transfecting MYCN overexpressed plasmid increased the PIF1 expression. We further found that downregulation of PIF1 expression by transfecting PIF1 siRNA (#1, #2) into NB cells, increased the number of apoptotic cells, inhibited the cell survival, decreased the ability of cell migration and induced a cell cycle arrest at G1 phase. These data indicated that PIF1, as a potential new target of MYCN, maybe a novel target for NB treatment.


Assuntos
Apoptose/genética , Movimento Celular/genética , DNA Helicases/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Biologia Computacional , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Regulação para Cima
8.
Proc Natl Acad Sci U S A ; 117(25): 14314-14321, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513739

RESUMO

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.


Assuntos
Eucromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Cromossomos , DNA Helicases , Replicação do DNA , Heterocromatina , Origem de Replicação/genética
9.
Nat Commun ; 11(1): 2781, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493900

RESUMO

Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1, in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity,  with  Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Alelos , Animais , Citocinas/metabolismo , DNA Helicases/deficiência , Diabetes Mellitus Tipo 1/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR3/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Linfócitos T Reguladores/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de Transcrição/deficiência
10.
Int J Nanomedicine ; 15: 3377-3389, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32494136

RESUMO

Background: Hepatitis C virus (HCV) infection is a major cause of hepatic diseases all over the world. This necessitates the need to discover novel anti-HCV drugs to overcome emerging drug resistance and liver complications. Purpose: Total extract and petroleum ether fraction of the marine sponge (Amphimedon spp.) were used for silver nanoparticle (SNP) synthesis to explore their HCV NS3 helicase- and protease-inhibitory potential. Methods: Characterization of the prepared SNPs was carried out with ultraviolet-visible spectroscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. The metabolomic profile of different Amphimedon fractions was assessed using liquid chromatography coupled with high-resolution mass spectrometry. Fourteen known compounds were isolated and their HCV helicase and protease activities assessed using in silico modeling of their interaction with both HCV protease and helicase enzymes to reveal their anti-HCV mechanism of action. In vitro anti-HCV activity against HCV NS3 helicase and protease was then conducted to validate the computation results and compared to that of the SNPs. Results: Transmission electron-microscopy analysis of NPs prepared from Amphimedon total extract and petroleum ether revealed particle sizes of 8.22-14.30 nm and 8.22-9.97 nm, and absorption bands at λmax of 450 and 415 nm, respectively. Metabolomic profiling revealed the richness of Amphimedon spp. with different phytochemical classes. Bioassay-guided isolation resulted in the isolation of 14 known compounds with anti-HCV activity, initially revealed by docking studies. In vitro anti-HCV NS3 helicase and protease assays of both isolated compounds and NPs further confirmed the computational results. Conclusion: Our findings indicate that Amphimedon, total extract, petroleum ether fraction, and derived NPs are promising biosources for providing anti-HCV drug candidates, with nakinadine B and 3,4-dihydro-6-hydroxymanzamine A the most potent anti-HCV agents, possessing good oral bioavailability and penetration power.


Assuntos
Simulação por Computador , DNA Helicases/antagonistas & inibidores , Química Verde , Metabolômica , Nanopartículas Metálicas/química , Poríferos/química , Prata/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Alcanos/química , Animais , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Oceano Índico , Nanopartículas Metálicas/ultraestrutura , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier
11.
Nucleic Acids Res ; 48(12): 6640-6653, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449930

RESUMO

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.


Assuntos
DNA Helicases/química , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Quadruplex G , Recombinases Rec A/química , Trifosfato de Adenosina/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligantes , Conformação de Ácido Nucleico , Recombinases Rec A/genética
12.
Nat Commun ; 11(1): 2639, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457312

RESUMO

Homologous recombination (HR) is important for error-free DNA double strand break repair and maintenance of genomic stability. However, upregulated HR is also used by cancer cells to promote therapeutic resistance. Therefore, inducing HR deficiency (HRD) is a viable strategy to sensitize HR proficient cancers to DNA targeted therapies in order to overcome therapeutic resistance. A bromodomain containing protein, BRD9, was previously reported to regulate chromatin remodeling and transcription. Here, we discover that following DNA damage, the bromodomain of BRD9 binds acetylated K515 on RAD54 and facilitates RAD54's interaction with RAD51, which is essential for HR. BRD9 is overexpressed in ovarian cancer and depleting BRD9 sensitizes cancer cells to olaparib and cisplatin. In addition, inhibitor of BRD9, I-BRD9, acts synergistically with olaparib in HR-proficient cancer cells. Overall, our results elucidate a role for BRD9 in HR and identify BRD9 as a potential therapeutic target to promote synthetic lethality and overcome chemoresistance.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/química , Rad51 Recombinase/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
13.
Nucleic Acids Res ; 48(12): 6980-6995, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453425

RESUMO

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one ß-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/ultraestrutura , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Conformação Proteica
14.
Nucleic Acids Res ; 48(11): 6326-6339, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374860

RESUMO

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Archaeoglobus fulgidus/química , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Soluções , Eletricidade Estática , Sulfolobus/química
15.
Nucleic Acids Res ; 48(12): 6855-6873, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32406909

RESUMO

Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into RNA granules reduces their translation, but it remains unclear whether RBPs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress adaptation in cancer. To study transcript partitioning under cell stress, we catalogued mRNAs enriched in prostate carcinoma PC-3 cell PSs, as defined by polysome fractionation and RNA sequencing (RNAseq), and compared them to mRNAs complexed with the known SG-nucleator protein, G3BP1, as defined by spatially-restricted enzymatic tagging and RNAseq. By comparing these compartments before and after short-term arsenite-induced oxidative stress, we identified three major categories of transcripts, namely those that were G3BP1-associated and PS-depleted, G3BP1-dissociated and PS-enriched, and G3BP1-associated but also PS-enriched. Oxidative stress profoundly altered the partitioning of transcripts between these compartments. Under arsenite stress, G3BP1-associated and PS-depleted transcripts correlated with reduced expression of encoded mitochondrial proteins, PS-enriched transcripts that disassociated from G3BP1 encoded cell cycle and cytoprotective proteins whose expression increased, while transcripts that were both G3BP1-associated and PS-enriched encoded proteins involved in diverse stress response pathways. Therefore, G3BP1 guides transcript partitioning to reprogram mRNA translation and support stress adaptation.


Assuntos
DNA Helicases/genética , Estresse Oxidativo/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Mensageiro/genética , Arsenitos/toxicidade , Carcinoma/genética , Carcinoma/metabolismo , Grânulos Citoplasmáticos/genética , Metabolismo Energético/genética , Humanos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas de Ligação a RNA/genética
16.
Nucleic Acids Res ; 48(11): 6120-6135, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421777

RESUMO

CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.


Assuntos
Adaptação Fisiológica/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , DNA/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/imunologia , Pareamento de Bases , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , DNA Helicases/metabolismo , Mutação , Hibridização de Ácido Nucleico , Plasmídeos/genética , Plasmídeos/metabolismo , Pyrococcus furiosus/metabolismo , RNA/genética , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(20): 10856-10864, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32371489

RESUMO

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.


Assuntos
DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Thermus/genética
18.
Nature ; 581(7807): 209-214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405004

RESUMO

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.


Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Organelas/química , Organelas/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Corpos Enovelados/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/deficiência , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transição de Fase , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/deficiência , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Ribossomos/química , Ribossomos/metabolismo
19.
PLoS One ; 15(5): e0233394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453735

RESUMO

Chromodomain helicase DNA-binding (CHD) chromatin remodelers regulate transcription and DNA repair. They govern cell-fate decisions during embryonic development and are often deregulated in human pathologies. Chd1-8 show upon germline disruption pronounced, often developmental lethal phenotypes. Here we show that contrary to Chd1-8 disruption, Chd9-/-animals are viable, fertile and display no developmental defects or disease predisposition. Germline deletion of Chd9 only moderately affects gene expression in tissues and derived cells, whereas acute depletion in human cancer cells elicits more robust changes suggesting that CHD9 is a highly context-dependent chromatin regulator that, surprisingly, is dispensable for mouse development.


Assuntos
DNA Helicases/genética , Transativadores/genética , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa , Humanos , Células K562 , Camundongos , Células-Tronco Embrionárias Murinas/citologia
20.
PLoS Genet ; 16(5): e1008787, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392243

RESUMO

During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5' UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Regiões 5' não Traduzidas , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Células HeLa , Recombinação Homóloga , Humanos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química
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