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1.
Science ; 372(6549): eabf6548, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34739333

RESUMO

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulum­associated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granule­specific interaction network, resulting in granule disassembly.


Assuntos
Proteínas Sanguíneas/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Resposta ao Choque Térmico , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteína com Valosina/metabolismo , Autofagia , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/genética , Poliubiquitina/metabolismo , Domínios Proteicos , Proteólise , RNA Helicases/química , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas Ubiquitinadas/química , Ubiquitinação
2.
PLoS Genet ; 17(10): e1009834, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34644293

RESUMO

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Células Germinativas/fisiologia , Células-Tronco/fisiologia , Animais , Autorrenovação Celular/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Feminino , Masculino , Transdução de Sinais/genética , Transcriptoma/genética
3.
Nat Commun ; 12(1): 5966, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645815

RESUMO

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.


Assuntos
Proteína BRCA2/genética , DNA Primase/genética , DNA de Neoplasias/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas de Manutenção de Minicromossomo/genética , Enzimas Multifuncionais/genética , Reparo de DNA por Recombinação , Proteína BRCA2/antagonistas & inibidores , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Primase/antagonistas & inibidores , DNA Primase/metabolismo , Replicação do DNA , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Proteínas de Manutenção de Minicromossomo/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Nat Commun ; 12(1): 5535, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545080

RESUMO

Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged ß-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Multimerização Proteica , Proteínas Virais/metabolismo , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/ultraestrutura
5.
Nat Commun ; 12(1): 5156, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526504

RESUMO

R-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the R-loop-specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease.


Assuntos
DNA Helicases/genética , DNA/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Enzimas Multifuncionais/genética , Processamento de Proteína Pós-Traducional , Proteostase/genética , RNA Helicases/genética , Tioléster Hidrolases/genética , Linhagem Celular , Senescência Celular/genética , DNA/química , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismo , Ubiquitinação
6.
Nature ; 598(7880): 368-372, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34526721

RESUMO

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.


Assuntos
Microscopia Crioeletrônica , Reparo do DNA , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Transcrição Genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação
7.
Nat Commun ; 12(1): 5404, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518526

RESUMO

Inactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca2+) release in these cancers. By restricting chromatin accessibility to ITPR3, encoding Ca2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca2+-dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , DNA Helicases/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias/metabolismo , Mutação , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/genética , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
Int J Biol Macromol ; 190: 636-648, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517025

RESUMO

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , COVID-19/tratamento farmacológico , Proteínas do Nucleocapsídeo de Coronavírus/química , DNA Helicases/química , Decitabina/química , Mesilato de Imatinib/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas de Ligação a RNA/química , SARS-CoV-2/química , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Decitabina/farmacologia , Sistemas de Liberação de Medicamentos , Genômica , Mesilato de Imatinib/farmacologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo
9.
Nat Commun ; 12(1): 5512, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535666

RESUMO

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Hibridização de Ácido Nucleico , Edição de RNA , RNA/metabolismo , Adenosina Desaminase/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Deleção de Genes , Genes Reporter , Instabilidade Genômica , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga/genética , Humanos , Enzimas Multifuncionais/metabolismo , Estabilidade Proteica , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Proteína de Replicação A/metabolismo
10.
Nat Commun ; 12(1): 5240, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475390

RESUMO

ß-actin is a crucial component of several chromatin remodeling complexes that control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes (PRCs). While previous work has shown that ß-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in ß-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of ß-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that ß-actin levels can induce changes in chromatin structure by affecting the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2. Our results show that changes in ß-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversible changes in 3D genome architecture. Our findings reveal that ß-actin-dependent chromatin remodeling plays a role in shaping the chromatin landscape and influences the regulation of genes involved in development and differentiation.


Assuntos
Actinas/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Actinas/genética , Animais , Cromatina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Fibroblastos , Dosagem de Genes , Técnicas de Inativação de Genes , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo
11.
Elife ; 102021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34486521

RESUMO

The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.


Assuntos
Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Transporte/genética , Microscopia Crioeletrônica , DNA Helicases/genética , DNA Helicases/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
12.
Cytogenet Genome Res ; 161(6-7): 285-296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34469893

RESUMO

Unique repetitive elements of the eukaryotic genome can be problematic for cellular DNA replication and transcription and pose a source of genomic instability. Human ribosomal DNA (rDNA) exists as repeating units clustered together on several chromosomes. Understanding the molecular mechanisms whereby rDNA interferes with normal genome homeostasis is the subject of this review. We discuss the instability of rDNA as a driver of senescence and the important roles of helicases to suppress its deleterious effects. The propensity of rDNA that is rich in guanine bases to form G-quadruplexes (G4) is discussed and evaluated in disease pathogenesis. Targeting G4 in the ribosomes and other chromosomal loci may represent a useful synthetic lethal approach to combating cancer.


Assuntos
DNA Ribossômico/genética , Quadruplex G , Genoma Humano/genética , Instabilidade Genômica , Neoplasias/genética , Sequências Repetitivas de Ácido Nucleico/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/genética , DNA Ribossômico/química , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/metabolismo
13.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576232

RESUMO

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.


Assuntos
Citocinese/fisiologia , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Neuroblastoma/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Interferência de RNA , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Centrossomo , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fuso Acromático
14.
Nucleic Acids Res ; 49(16): 9066-9076, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34365508

RESUMO

Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone octamers and forms nucleosomes, translocases inevitably encounter nucleosomes. A previous study has shown that a nucleosome repositions downstream when a translocase collides with the nucleosome. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identified the lane-switch mechanism for downstream repositioning with molecular dynamics simulations and validated it with restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have broad implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.


Assuntos
Montagem e Desmontagem da Cromatina , Simulação de Dinâmica Molecular , Nucleossomos/química , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo
15.
Metab Eng ; 67: 396-402, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34411701

RESUMO

CRISPR-enabled deaminase base editing has become a powerful tool for precisely editing nucleotides on the chromosome. In this study DNA helicases, such as Escherichia coli DnaB, were fused to activation-induced cytidine deaminase (AID) to form enzyme complexes which randomly introduces edited bases throughout the chromosome. DnaB-AID was found to increase 2.5 × 103 fold relative to the mutagenesis frequency of wildtype. 97.9% of these edits were observed on the leading strand during DNA replication suggesting deamination to be highly coordinated with DNA replication. Using DnaB-AID, a 371.4% increase in ß-carotene production was obtained following four rounds of editing. In Saccharomyces cerevisiae Helicase-AID was constructed by fusing AID to one of the subunits of eukaryotic helicase Mcm2-7 complex, MCM5. Using MCM5-AID, the average editing efficiency of five strains was 2.1 ± 0.4 × 103 fold higher than the native genomic mutation rate. MCM5-AID was able to improve ß-carotene production of S. cerevisiae 4742crt by 75.4% following eight rounds of editing. The S. cerevisiae MCM5-AID technique is the first biological tool for generating and accumulating single base mutations in eukaryotic chromosomes. Since the helicase complex is highly conservative in all eukaryotes, Helicase-AID could be adapted for various applications and research in all eukaryotic cells.


Assuntos
DNA Helicases , Saccharomyces cerevisiae , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Helicases/metabolismo , Genoma , Genômica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
Biophys Chem ; 278: 106677, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34428682

RESUMO

The SARS-CoV-2 pandemic has accelerated the study of existing drugs. The mixture of homologs called ivermectin (avermectin-B1a [HB1a] + avermectin-B1b [HB1b]) has shown antiviral activity against SARS-CoV-2 in vitro. However, there are few reports on the behavior of each homolog. We investigated the interaction of each homolog with promising targets of interest associated with SARS-CoV-2 infection from a biophysical and computational-chemistry perspective using docking and molecular dynamics. We observed a differential behavior for each homolog, with an affinity of HB1b for viral structures, and of HB1a for host structures considered. The induced disturbances were differential and influenced by the hydrophobicity of each homolog and of the binding pockets. We present the first comparative analysis of the potential theoretical inhibitory effect of both avermectins on biomolecules associated with COVID-19, and suggest that ivermectin through its homologs, has a multiobjective behavior.


Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , DNA Helicases/antagonistas & inibidores , Ivermectina/análogos & derivados , alfa Carioferinas/antagonistas & inibidores , beta Carioferinas/antagonistas & inibidores , Animais , Antivirais/farmacologia , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/virologia , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Humanos , Ivermectina/química , Ivermectina/farmacologia , Cinética , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Termodinâmica , alfa Carioferinas/química , alfa Carioferinas/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismo
17.
Trends Neurosci ; 44(10): 765-766, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34429216

RESUMO

A recent study by Gwon et al. identified context-specific ubiquitination of G3BP1 as critical for stress granule disassembly via VCP and the adaptor FAF2. This study provides new insights into stress granule dynamics, with potential implications for neurodegenerative disease.


Assuntos
Esclerose Amiotrófica Lateral , Doenças Neurodegenerativas , DNA Helicases/metabolismo , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA
18.
Biochemistry ; 60(37): 2795-2809, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34464102

RESUMO

The geminivirus replication protein, Rep, has long been recognized as a high-value target for control of geminivirus infections as this protein is highly conserved and essential for viral replication and proliferation. In addition, inhibition of viral replication has been pursued through various antiviral strategies with varying degrees of success, including inhibitory peptides that target Rep. While much effort has centered around sequence characterization of the Rep protein and inhibitory peptides, detailed structural analysis has been missing. This study computationally investigated the presence of common structural features within these inhibitory peptides and if these features could inform if a particular peptide will bind Rep and/or interfere with viral replication. Molecular dynamics simulations of the inhibitory peptide library showed that simply possessing stable structural features does not inform interference of viral replication regardless of the binding of Rep. Additionally, nearly all known Rep inhibitory peptides sample a conserved ß-sheet structural motif, possibly informing structure-function relationships in binding Rep. In particular, two peptides (A22 and A64) characterized by this structural motif were computationally docked against a wide variety of geminivirus Rep proteins to determine a mechanism of action. Computational docking revealed these peptides utilize a common Rep protein sequence motif for binding, HHN-x1/2-Q. The results identified residues in both Rep and the inhibitory peptides that play a significant role in the interaction, establishing the foundation for a rational structure-based design approach for the construction of both broadly reactive and geminivirus species-specific inhibitors.


Assuntos
Geminiviridae/enzimologia , Geminiviridae/metabolismo , Replicação Viral/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , DNA Helicases/metabolismo , DNA Viral/metabolismo , Geminiviridae/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Replicação Viral/genética
19.
Nat Commun ; 12(1): 5123, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446700

RESUMO

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.


Assuntos
Trifosfato de Adenosina/metabolismo , Cromatina/metabolismo , DNA Helicases/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética
20.
Nat Commun ; 12(1): 4451, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294712

RESUMO

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.


Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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