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1.
Nat Commun ; 11(1): 3940, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769985

RESUMO

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo
2.
Nat Commun ; 11(1): 3143, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561719

RESUMO

Topoisomerase 3ß (Top3ß) is the only dual-activity topoisomerase in animals that can change topology for both DNA and RNA, and facilitate transcription on DNA and translation on mRNAs. Top3ß mutations have been linked to schizophrenia, autism, epilepsy, and cognitive impairment. Here we show that Top3ß knockout mice exhibit behavioural phenotypes related to psychiatric disorders and cognitive impairment. The mice also display impairments in hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many with known neuronal functions. Our data suggest that Top3ß is essential for normal brain function, and that defective neuronal activity-dependent transcription may be a mechanism by which Top3ß deletion causes cognitive impairment and psychiatric disorders.


Assuntos
Disfunção Cognitiva/genética , DNA Topoisomerases Tipo I/genética , Transtornos Mentais/genética , Neurogênese/genética , Plasticidade Neuronal/genética , Animais , Técnicas de Observação do Comportamento , Comportamento Animal , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Feminino , Hipocampo/citologia , Hipocampo/diagnóstico por imagem , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Humanos , Imagem por Ressonância Magnética , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/patologia , Camundongos , Camundongos Knockout , Neurônios/patologia , Técnicas Estereotáxicas , Potenciais Sinápticos/genética , Transcrição Genética/fisiologia
3.
Nat Commun ; 11(1): 1962, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327659

RESUMO

Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD+) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.


Assuntos
DNA Topoisomerases Tipo I/deficiência , Instabilidade Genômica , Doenças Neurodegenerativas/enzimologia , Neurônios/enzimologia , Animais , Apoptose/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Dano ao DNA , DNA Topoisomerases Tipo I/genética , Hipocampo/enzimologia , Hipocampo/patologia , Inflamação , Camundongos , Camundongos Knockout , Mortalidade Prematura , Atividade Motora , Mutação , NAD/administração & dosagem , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Poli(ADP-Ribose) Polimerase-1/metabolismo
4.
Nucleic Acids Res ; 48(8): 4448-4462, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32232337

RESUMO

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.


Assuntos
DNA Topoisomerases Tipo I/química , DNA de Cadeia Simples/metabolismo , Mycobacterium smegmatis/enzimologia , Cristalografia por Raios X , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Modelos Moleculares , Domínios Proteicos , Deleção de Sequência
5.
Open Biol ; 10(2): 190228, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32019477

RESUMO

Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3ß) with similarity to the mammalian topoisomerase IIIß. We found that TOP3ß was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3ß can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3ß can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3ß increased expression of cwp1-3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3ß. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3ß gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3ß may be functionally conserved, and involved in inducing Giardia cyst formation.


Assuntos
DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Perfilação da Expressão Gênica/métodos , Giardia lamblia/fisiologia , Domínio Catalítico , Parede Celular/metabolismo , DNA Topoisomerases Tipo I/química , Regulação da Expressão Gênica , Giardia lamblia/enzimologia , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transativadores/metabolismo , Regulação para Cima
6.
Nat Commun ; 11(1): 908, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075966

RESUMO

Cyclic cGMP-AMP synthase (cGAS) is a pattern recognition cytosolic DNA sensor that is essential for cellular senescence. cGAS promotes inflammatory senescence-associated secretory phenotype (SASP) through recognizing cytoplasmic chromatin during senescence. cGAS-mediated inflammation is essential for the antitumor effects of immune checkpoint blockade. However, the mechanism by which cGAS recognizes cytoplasmic chromatin is unknown. Here we show that topoisomerase 1-DNA covalent cleavage complex (TOP1cc) is both necessary and sufficient for cGAS-mediated cytoplasmic chromatin recognition and SASP during senescence. TOP1cc localizes to cytoplasmic chromatin and TOP1 interacts with cGAS to enhance the binding of cGAS to DNA. Retention of TOP1cc to cytoplasmic chromatin depends on its stabilization by the chromatin architecture protein HMGB2. Functionally, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Together, these findings establish a HMGB2-TOP1cc-cGAS axis that enables cytoplasmic chromatin recognition and response to immune checkpoint blockade.


Assuntos
Senescência Celular/imunologia , DNA Topoisomerases Tipo I/metabolismo , Proteína HMGB2/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Antígeno B7-H1/imunologia , Linhagem Celular , Cromatina/imunologia , Cromatina/metabolismo , Citosol/imunologia , Citosol/metabolismo , DNA/imunologia , DNA/metabolismo , Dano ao DNA/imunologia , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Proteína HMGB2/genética , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neoplasias/imunologia , Nucleotidiltransferases/genética , Ligação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Cell ; 77(5): 1066-1079.e9, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31902667

RESUMO

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1 wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S phase-dependent manner to assist in the eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among the potential Ddi1 targets, we found the core component of Pol II and show that its genotoxin-induced degradation is impaired in ddi1. We propose that the Ddi1 protease contributes to DPC proteolysis.


Assuntos
Dano ao DNA , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Animais , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteólise , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9 , Spodoptera , Transcrição Genética
8.
Methods Mol Biol ; 2102: 195-210, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989556

RESUMO

RNA interference (RNAi) is a powerful approach for inhibiting gene expression and its wide applications have expanded our understanding of gene functions. Short hairpin RNAs (shRNAs) are artificially synthesized RNA molecules used to mediate RNAi. The expression of shRNA in cells can be achieved by using plasmids or viral/bacterial vectors. The use of viral vectors to carry and deliver shRNA shows many advantages, including the ability to overcome the difficulty of transfecting certain cell types, the capability to establish stable cell lines via antibiotics selection or fluorescence-activated cell sorting, and the option to control temporally shRNA expression using inducible promoters. In this chapter, we introduce a gene silencing method utilizing a lentivirus-based inducible shRNA system. Using the human topoisomerase (TOP) gene as an example, we describe a procedure to generate stable HepG2 cells showing inducible suppression of TOP1. In addition, a procedure for assessing the efficiency of gene silencing is described in detail.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Lentivirus/genética , Interferência de RNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Vetores Genéticos , Células Hep G2 , Humanos , RNA Interferente Pequeno , Fluxo de Trabalho
9.
Nucleic Acids Res ; 48(2): 761-769, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31777935

RESUMO

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.


Assuntos
Dióxido de Carbono/metabolismo , DNA Topoisomerases Tipo I/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Biomassa , Dióxido de Carbono/química , DNA Topoisomerases Tipo I/química , Farmacorresistência Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Evolução Molecular , Duplicação Gênica/genética , Genótipo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Mutação , Mutação Puntual/genética
10.
Nature ; 576(7786): 274-280, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31802000

RESUMO

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.


Assuntos
MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , RNA Helicases DEAD-box/genética , DNA Topoisomerases Tipo I/genética , Humanos , Mutação , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Polimorfismo de Nucleotídeo Único , Recidiva , Ribonuclease III/genética
11.
BMC Cancer ; 19(1): 1158, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783818

RESUMO

BACKGROUND: Camptothecin (CPT) and its derivatives are currently used as second- or third-line treatment for patients with endocrine-resistant breast cancer (BC). These drugs convert nuclear enzyme DNA topoisomerase I (TOP1) to a cell poison with the potential to damage DNA by increasing the half-life of TOP1-DNA cleavage complexes (TOP1cc), ultimately resulting in cell death. In small and non-randomized trials for BC, researchers have observed extensive variation in CPT response rates, ranging from 14 to 64%. This variability may be due to the absence of reliable selective parameters for patient stratification. BC cell lines may serve as feasible models for generation of functional criteria that may be used to predict drug sensitivity for patient stratification and, thus, lead to more appropriate applications of CPT in clinical trials. However, no study published to date has included a comparison of multiple relevant parameters and CPT response across cell lines corresponding to specific BC subtypes. METHOD: We evaluated the levels and possible associations of seven parameters including the status of the TOP1 gene (i.e. amplification), TOP1 protein expression level, TOP1 activity and CPT susceptibility, activity of the tyrosyl-DNA phosphodiesterase 1 (TDP1), the cellular CPT response and the cellular growth rate across a representative panel of BC cell lines, which exemplifies three major BC subtypes: Luminal, HER2 and TNBC. RESULTS: In all BC cell lines analyzed (without regard to subtype classification), we observed a significant overall correlation between growth rate and CPT response. In cell lines derived from Luminal and HER2 subtypes, we observed a correlation between TOP1 gene copy number, TOP1 activity, and CPT response, although the data were too limited for statistical analyses. In cell lines representing Luminal and TNBC subtypes, we observed a direct correlation between TOP1 protein abundancy and levels of enzymatic activity. In all three subtypes (Luminal, HER2, and TNBC), TOP1 exhibits approximately the same susceptibility to CPT. Of the three subtypes examined, the TNBC-like cell lines exhibited the highest CPT sensitivity and were characterized by the fastest growth rate. This indicates that breast tumors belonging to the TNBC subtype, may benefit from treatment with CPT derivatives. CONCLUSION: TOP1 activity is not a marker for CPT sensitivity in breast cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/enzimologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , Feminino , Dosagem de Genes , Expressão Gênica , Humanos , Diester Fosfórico Hidrolases/metabolismo
12.
Anal Chem ; 91(23): 14927-14935, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31710202

RESUMO

Cancer drug resistance mechanisms such as tumor heterogeneity and adaptable feedback loops are prevalent issues facing cancer therapy development. Drug resistance can be unique to a cancer type and, most importantly, to each individual cancer patient. Consequently, testing different dosages and therapeutics directly on each individual patient sample (i.e., tumor and cancer cells) has compelling advantages compared to large scale in vitro drug testing and is a step toward personalized drug selection and effective treatment development. Recently, microfluidic-based chemo-sensitivity assays on patient biopsies have been proposed. Despite their novelty, these platforms usually rely on optical labels, optical equipment, or complex microfabricated channel geometries and structures. In this work, we proposed a novel lab on a chip platform capable of real-time and continuous screening of drug efficacy on (cancer) cell subpopulations without the need of labels or bulky readout optical equipment. In this platform, several label-free and rapid techniques have been implemented for the precise capturing of cells of interest in parallel with the real-time measurement and characterization of the effectiveness of candidate therapeutic agents. To demonstrate the utility of the platform, the effect of an apoptotic inducer, topoisomerase I inhibitor, 7-ethyl-10-hydrocamptothecin (SN38) on human colorectal carcinoma cancer cells (HCT 116) was used as a study model. Additionally, electrical results were optically verified to examine the continuous measurements of the biological mechanisms, specifically, apoptosis and necrosis, during therapeutic agent characterizations. The proposed device is a versatile platform which can also be easily redesigned for the automated and arrayed analysis of cell-drug interaction down to the single cell level. Our platform is another step toward enabling the personalized screening of drug efficacy on individual patients' samples that potentially leads to a better understanding of drug resistance and the optimization of patients' treatments.


Assuntos
DNA Topoisomerases Tipo I/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Irinotecano/farmacologia , Dispositivos Lab-On-A-Chip , Inibidores da Topoisomerase I/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , DNA Topoisomerases Tipo I/metabolismo , Monitoramento de Medicamentos/métodos , Impedância Elétrica , Células HCT116 , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Medicina de Precisão
13.
Genes (Basel) ; 10(11)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698852

RESUMO

The conserved eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1) removes a diverse array of adducts from the end of DNA strand breaks. Tdp1 specifically catalyzes the hydrolysis of phosphodiester linked DNA-adducts. These DNA lesions range from damaged nucleotides to peptide-DNA adducts to protein-DNA covalent complexes and are products of endogenously or exogenously induced insults or simply failed reaction products. These adducts include DNA inserted ribonucleotides and non-conventional nucleotides, as well as covalent reaction intermediates of DNA topoisomerases with DNA and a Tdp1-DNA adduct in trans. This implies that Tdp1 plays a role in maintaining genome stability and cellular homeostasis. Dysregulation of Tdp1 protein levels or catalysis shifts the equilibrium to genome instability and is associated with driving human pathologies such as cancer and neurodegeneration. In this review, we highlight the function of the N-terminal domain of Tdp1. This domain is understudied, structurally unresolved, and the least conserved in amino acid sequence and length compared to the rest of the enzyme. However, over time it emerged that the N-terminal domain was post-translationally modified by, among others, phosphorylation, SUMOylation, and Ubiquitinoylation, which regulate Tdp1 protein interactions with other DNA repair associated proteins, cellular localization, and Tdp1 protein stability.


Assuntos
Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Catálise , DNA/química , Dano ao DNA , Reparo do DNA , DNA Topoisomerases Tipo I/genética , Humanos , Hidrólise
14.
Mol Immunol ; 116: 63-72, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622795

RESUMO

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.


Assuntos
Citidina Desaminase/genética , DNA Topoisomerases Tipo I/genética , Genes de Imunoglobulinas/genética , Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Hipermutação Somática de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linhagem Celular , Núcleo Celular/genética , Galinhas/genética , Switching de Imunoglobulina , Imunoprecipitação/métodos , Camundongos , Isoformas de Proteínas/genética
15.
Mol Biol Evol ; 36(12): 2737-2747, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504731

RESUMO

Reverse gyrase (RG) is the only protein found ubiquitously in hyperthermophilic organisms, but absent from mesophiles. As such, its simple presence or absence allows us to deduce information about the optimal growth temperature of long-extinct organisms, even as far as the last universal common ancestor of extant life (LUCA). The growth environment and gene content of the LUCA has long been a source of debate in which RG often features. In an attempt to settle this debate, we carried out an exhaustive search for RG proteins, generating the largest RG data set to date. Comprising 376 sequences, our data set allows for phylogenetic reconstructions of RG with unprecedented size and detail. These RG phylogenies are strikingly different from those of universal proteins inferred to be present in the LUCA, even when using the same set of species. Unlike such proteins, RG does not form monophyletic archaeal and bacterial clades, suggesting RG emergence after the formation of these domains, and/or significant horizontal gene transfer. Additionally, the branch lengths separating archaeal and bacterial groups are very short, inconsistent with the tempo of evolution from the time of the LUCA. Despite this, phylogenies limited to archaeal RG resolve most archaeal phyla, suggesting predominantly vertical evolution since the time of the last archaeal ancestor. In contrast, bacterial RG indicates emergence after the last bacterial ancestor followed by significant horizontal transfer. Taken together, these results suggest a nonhyperthermophilic LUCA and bacterial ancestor, with hyperthermophily emerging early in the evolution of the archaeal and bacterial domains.


Assuntos
DNA Topoisomerases Tipo I/genética , Evolução Molecular , Origem da Vida , Filogenia , Transferência Genética Horizontal
16.
Cell Host Microbe ; 26(3): 369-384.e8, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31513772

RESUMO

Pathogen pattern recognition receptors (PRRs) trigger innate immune responses to invading pathogens. All known PRRs for viral RNA have extranuclear localization. However, for many viruses, replication generates dsRNA in the nucleus. Here, we show that the nuclear matrix protein SAFA (also known as HnRNPU) functions as a nuclear viral dsRNA sensor for both DNA and RNA viruses. Upon recognition of viral dsRNA, SAFA oligomerizes and activates the enhancers of antiviral genes, including IFNB1. Moreover, SAFA is required for the activation of super-enhancers, which direct vigorous immune gene transcription to establish the antiviral state. Myeloid-specific SAFA-deficient mice were more susceptible to lethal HSV-1 and VSV infection, with decreased type I IFNs. Thus, SAFA functions as a nuclear viral RNA sensor and trans-activator to bridge innate sensing with chromatin remodeling and potentiate robust antiviral responses.


Assuntos
Antivirais/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/imunologia , Proteínas Associadas à Matriz Nuclear/imunologia , RNA Viral/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Adenosina Trifosfatases/genética , Animais , Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo I/genética , Vírus de DNA , Células HEK293 , Células HeLa , Herpesvirus Humano 1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon , Fator Regulador 7 de Interferon , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Serina-Treonina Quinases , Vírus de RNA , RNA de Cadeia Dupla , Vírus
17.
Cell Cycle ; 18(19): 2377-2384, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31345095

RESUMO

Mitochondria contain their own genome (mtDNA), encoding 13 proteins of the enzyme complexes of the oxidative phosphorylation. Synthesis of these 13 mitochondrial proteins requires a specific translation machinery, the mitoribosomes whose RNA components are encoded by the mtDNA, whereas more than 80 proteins are encoded by nuclear genes. It has been well established that mitochondrial topoisomerase I (TOP1MT) is important for mtDNA integrity and mitochondrial transcription as it prevents excessive mtDNA negative supercoiling and releases topological stress during mtDNA replication and transcription. We recently showed that TOP1MT also supports mitochondrial protein synthesis, and thus is critical for promoting tumor growth. Impaired mitochondrial protein synthesis leads to activation of the mitonuclear stress response through the transcription factor ATF4, and induces cytoprotective genes in order to prevent mitochondrial and cellular dysfunction. In this perspective, we highlight the novel role of TOP1MT in mitochondrial protein synthesis and as potential target for chemotherapy.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas , Fator 4 Ativador da Transcrição/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proliferação de Células/genética , Replicação do DNA/genética , DNA Topoisomerases Tipo I/genética , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Genética
18.
BMC Mol Cell Biol ; 20(1): 26, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319794

RESUMO

BACKGROUND: The processes of DNA supercoiling and transcription are interdependent because the movement of a transcription elongation complex simultaneously induces under- and overwinding of the DNA duplex and because the initiation, elongation and termination steps of transcription are all sensitive to the topological state of the DNA. RESULTS: Policing of the local and global supercoiling of DNA by topoisomerases helps to sustain the major DNA-based transactions by eliminating barriers to the movement of transcription complexes and replisomes. Recent data from whole-genome and single-molecule studies have provided new insights into how interactions between transcription and the supercoiling of DNA influence the architecture of the chromosome and how they create cell-to-cell diversity at the level of gene expression through transcription bursting. CONCLUSIONS: These insights into fundamental molecular processes reveal mechanisms by which bacteria can prevail in unpredictable and often hostile environments by becoming unpredictable themselves.


Assuntos
DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Transcrição Genética , Ativação Transcricional/genética , Sítios de Ligação , Cromossomos Bacterianos/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/genética , DNA de Cadeia Simples/genética , Escherichia coli/genética , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Ligação Proteica
19.
DNA Repair (Amst) ; 84: 102641, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31311768

RESUMO

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Taxa de Mutação , Ribonucleotídeos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Polimerase Dirigida por DNA/metabolismo , Repetições de Dinucleotídeos , Deleção de Genes , Instabilidade Genômica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Repetições de Trinucleotídeos
20.
Mol Biotechnol ; 61(8): 622-630, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31165966

RESUMO

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 µg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.


Assuntos
DNA Topoisomerases Tipo I/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Vírus Vaccinia , Proteínas Virais/isolamento & purificação , Animais , Baculoviridae/genética , Bombyx/metabolismo , Bombyx/virologia , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Magnésio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus Vaccinia/enzimologia , Vírus Vaccinia/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
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