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1.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
2.
Acta Crystallogr D Struct Biol ; 75(Pt 5): 498-504, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31063152

RESUMO

Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53 Šresolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α-helices, five ß-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.


Assuntos
Encéfalo/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Esclerose Múltipla/metabolismo , Plasmídeos/isolamento & purificação , Plasmídeos/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência
4.
Nat Commun ; 10(1): 1720, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979890

RESUMO

ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.


Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/análise , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Animais , Tampões (Química) , DNA Helicases/química , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Humanos , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Xenopus
5.
Nat Biotechnol ; 37(6): 651-656, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011178

RESUMO

Nanopore DNA sequencing is limited by low base-calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage from 100 to 200 mV, with a frequency of 200 Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.7 ± 0.5% with a constant driving voltage improves to 79.3 ± 0.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.


Assuntos
DNA Helicases/genética , DNA/genética , Nanoporos , Porinas/genética , Algoritmos , DNA/isolamento & purificação , DNA Helicases/química , Mycobacterium smegmatis/genética , Porinas/química , Análise de Sequência de DNA/métodos
6.
Plant Mol Biol ; 100(1-2): 133-149, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30843130

RESUMO

KEY MESSAGE: The OsPLS2 locus was isolated and cloned by map-based cloning that encodes a Upf1-like helicase. Disruption of OsPLS2 accelerated light-dependent leaf senescence in the rice mutant of ospls2. Leaf senescence is a very complex physiological process controlled by both genetic and environmental factors, however its underlying molecular mechanisms remain elusive. In this study, we report a novel Oryza sativa premature leaf senescence mutant (ospls2). Through map-based cloning, a G-to-A substitution was determined at the 1st nucleotide of the 13th intron in the OsPLS2 gene that encodes a Upf1-like helicase. This mutation prompts aberrant splicing of OsPLS2 messenger and consequent disruption of its full-length protein translation, suggesting a negative role of OsPLS2 in regulating leaf senescence. Wild-type rice accordingly displayed a progressive drop of OsPSL2 protein levels with age-dependent leaf senescence. Shading and light filtration studies showed that the ospls2 phenotype, which was characteristic of photo-oxidative stress and reactive oxygen species (ROS) accumulation, was an effect of irritation by light. When continuously exposed to far-red light, exogenous H2O2 and/or abscisic acid (ABA), the ospls2 mutant sustained hypersensitive leaf senescence. In consistence, light and ROS signal pathways in ospls2 were activated by down-regulation of phytochrome genes, and up-regulation of PHYTOCHROME-INTERACTING FACTORS (PIFs) and WRKY genes, all promoting leaf senescence. Together, these data indicated that OsPLS2 played an essential role in leaf senescence and its disruption triggered light-dependent leaf senescence in rice.


Assuntos
DNA Helicases/genética , Genes de Plantas , Luz , Oryza/crescimento & desenvolvimento , Oryza/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Antioxidantes/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oryza/enzimologia , Oryza/efeitos da radiação , Fenótipo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Folhas de Planta/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
7.
Nature ; 567(7747): 267-272, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842657

RESUMO

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , DNA/química , DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA/biossíntese , Replicação do DNA , Feminino , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitinação , Xenopus
8.
Nucleic Acids Res ; 47(7): 3784-3794, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30753618

RESUMO

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Ubiquitina/química , Ubiquitinas/química , Fatores de Transcrição Winged-Helix/química , Sequência de Aminoácidos/genética , Sobrevivência Celular , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Conformação Proteica em alfa-Hélice/genética , Ubiquitina/genética , Ubiquitinas/genética , Raios Ultravioleta , Fatores de Transcrição Winged-Helix/genética
9.
J Biol Chem ; 294(9): 3294-3310, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30647132

RESUMO

The Snf2 proteins, comprising 53 different enzymes in humans, belong to the SF2 family. Many Snf2 enzymes possess chromatin-remodeling activity, requiring a functional ATPase domain consisting of conserved motifs named Q and I-VII. These motifs form two recA-like domains, creating an ATP-binding pocket. Little is known about the function of the conserved motifs in chromatin-remodeling enzymes. Here, we characterized the function of the Q and I (Walker I) motifs in hBRG1 (SMARCA4). The motifs are in close proximity to the bound ATP, suggesting a role in nucleotide binding and/or hydrolysis. Unexpectedly, when substituting the conserved residues Gln758 (Q motif) or Lys785 (I motif) of both motifs, all variants still bound ATP and exhibited basal ATPase activity similar to that of wildtype BRG1 (wtBRG1). However, all mutants lost the nucleosome-dependent stimulation of the ATPase domain. Their chromatin-remodeling rates were impaired accordingly, but nucleosome binding was retained and still comparable with that of wtBRG1. Interestingly, a cancer-relevant substitution, L754F (Q motif), displayed defects similar to the Gln758 variant(s), arguing for a comparable loss of function. Because we excluded a mutual interference of ATP and nucleosome binding, we postulate that both motifs stimulate the ATPase and chromatin-remodeling activities upon binding of BRG1 to nucleosomes, probably via allosteric mechanisms. Furthermore, mutations of both motifs similarly affect the enzymatic functionality of BRG1 in vitro and in living cells. Of note, in BRG1-deficient H1299 cells, exogenously expressed wtBRG1, but not BRG1 Q758A and BRG1 K785R, exhibited a tumor suppressor-like function.


Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , DNA Helicases/genética , Humanos , Modelos Moleculares , Mutação , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Fatores de Transcrição/genética
10.
Science ; 363(6429)2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30679383

RESUMO

Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo-electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A ß hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.


Assuntos
Bacteriófago T7/enzimologia , Bacteriófago T7/fisiologia , DNA Helicases/química , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Proteínas Virais/química , Replicação Viral , Microscopia Crioeletrônica , Domínios Proteicos
11.
J Biol Chem ; 294(8): 2801-2814, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30593500

RESUMO

DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a ß-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the ß-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA ß-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted ß-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/química , DNA/genética , DNA Helicases/química , DNA Helicases/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformação de Ácido Nucleico , Conformação Proteica
12.
BMC Mol Biol ; 19(1): 12, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30563453

RESUMO

BACKGROUND: DNA replication requires contributions from various proteins, such as DNA helicases; in mitochondria Twinkle is important for maintaining and replicating mitochondrial DNA. Twinkle helicases are predicted to also possess primase activity, as has been shown in plants; however this activity appears to have been lost in metazoans. Given this, the study of Twinkle in other organisms is required to better understand the evolution of this family and the roles it performs within mitochondria. RESULTS: Here we describe the characterization of a Twinkle homologue, Twm1, in the amoeba Dictyostelium discoideum, a model organism for mitochondrial genetics and disease. We show that Twm1 is important for mitochondrial function as it maintains mitochondrial DNA copy number in vivo. Twm1 is a helicase which unwinds DNA resembling open forks, although it can act upon substrates with a single 3' overhang, albeit less efficiently. Furthermore, unlike human Twinkle, Twm1 has primase activity in vitro. Finally, using a novel in bacterio approach, we demonstrated that Twm1 promotes DNA replication. CONCLUSIONS: We conclude that Twm1 is a replicative mitochondrial DNA helicase which is capable of priming DNA for replication. Our results also suggest that non-metazoan Twinkle could function in the initiation of mitochondrial DNA replication. While further work is required, this study has illuminated several alternative processes of mitochondrial DNA maintenance which might also be performed by the Twinkle family of helicases.


Assuntos
DNA Helicases/metabolismo , DNA Primase/metabolismo , Replicação do DNA , Dictyostelium/genética , Dictyostelium/metabolismo , Proteínas Mitocondriais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , DNA Helicases/química , DNA Helicases/genética , DNA Primase/química , DNA Primase/genética , DNA Mitocondrial , Dosagem de Genes , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Antissenso/genética , Especificidade por Substrato
13.
Proc Natl Acad Sci U S A ; 115(39): E9075-E9084, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30201718

RESUMO

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA's structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.


Assuntos
DNA Helicases/química , Replicação do DNA , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Modelos Biológicos , Cristalografia por Raios X , Domínios Proteicos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
14.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976661

RESUMO

Two replicase (Rep) proteins, Rep and Rep', are encoded by porcine circovirus (PCV) ORF1; Rep is a full ORF1 transcript, and Rep' is a truncated transcript generated by splicing. These two proteins are crucial for the rolling-circle replication (RCR) of PCV. The N-terminal sequences of Rep and Rep' are identical and interact to form homo- or heterodimers. The three types of dimers perform different functions during replication. A structural examination of the interfacing termini has not been performed. In this study, a crystal structure of dimerized Rep protein N termini was resolved at 2.7 Å. The dimerized protein was maintained by nine intermolecular hydrogen bonds and 15 pairs of hydrophobic interactions. The amino acid residue Ile37 participates in 11 of the hydrophobic interactions, mostly with its side chain. To find the predominant sites for protein dimerization and virus replication, a series of mutant proteins and virus replicons were generated by alanine substitution. Of all the single amino acid substitutions, the mutation at Ile37 showed the greatest effect on protein dimerization and virus replication. A double mutation at Leu35 and Ile37 almost eliminated protein dimerization and had the greatest negative effect on virus replication. These studies demonstrate that Leu35 and Ile37 are the most important residues for protein dimerization and are crucial for virus replication. Our results also show that PCV replication can be decreased by disrupting the dimerization of Rep or Rep' at the N terminus, suggesting that the structural interface responsible for dimerization offers a promising antiviral target.IMPORTANCE Porcine circovirus type 2 (PCV2) is one of the most economically damaging pathogens affecting the swine industry. Although vaccines have been available for more than 10 years, the virus still remains prevalent. More effective strategies for disease prevention are clearly required. The Rep and Rep' proteins of the virus have identical N-terminal regions that interact with each other, allowing the formation of homo- or heterodimers. The heterodimer has crucial functions during different stages of viral replication. Here, we resolved the crystal structure of the Rep (Rep') dimerization domain. The individual residues involved in the intermolecular interaction were visualized in the protein structure, and several interactions were verified by mutant analysis. Our studies show that disrupting the interaction decreases viral replication, thus revealing a new target for the design of antiviral agents.


Assuntos
Circovirus/enzimologia , DNA Helicases/química , Dimerização , Proteínas Virais/química , Substituição de Aminoácidos , Animais , Circovirus/química , Cristalização , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Viral/genética , Mutação , Suínos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
15.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976672

RESUMO

Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivoIMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.


Assuntos
DNA Helicases/química , DNA Helicases/fisiologia , DNA Primase/química , DNA Primase/fisiologia , Herpesvirus Humano 1/patogenicidade , Proteínas Virais/química , Proteínas Virais/fisiologia , Liberação de Vírus , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Cercopithecus aethiops , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Primase/genética , DNA Primase/isolamento & purificação , Olho/virologia , Células HEK293 , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases , Células Vero , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Vírion/fisiologia , Virulência , Montagem de Vírus
16.
J Mol Model ; 24(7): 176, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29943199

RESUMO

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.


Assuntos
DNA Helicases/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , DNA Helicases/genética , DNA Helicases/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Ativação Enzimática , Hidrólise , Simulação de Dinâmica Molecular , Complexos Multienzimáticos/química , Mutação , Análise de Componente Principal , Conformação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
17.
Proc Natl Acad Sci U S A ; 115(26): 6727-6732, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891665

RESUMO

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.


Assuntos
DNA Helicases/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/fisiologia , Miócitos Cardíacos/fisiologia , Sarcômeros/fisiologia , Animais , DNA Helicases/química , DNA Helicases/deficiência , Feminino , Técnicas de Silenciamento de Genes , Genes Letais , Coração/diagnóstico por imagem , Coração/embriologia , Cardiopatias Congênitas/diagnóstico por imagem , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/patologia , Masculino , Camundongos , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Miofibrilas/metabolismo , Miofibrilas/patologia , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Sarcômeros/ultraestrutura , Transcrição Genética , Ultrassonografia Pré-Natal
18.
Science ; 361(6397)2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29880725

RESUMO

Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.


Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Quebras de DNA de Cadeia Simples , Fragmentação do DNA , DNA Helicases/química , DNA/química , RNA Guia/química , Actinobacteria/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Microscopia Crioeletrônica , DNA/genética , DNA Helicases/genética , Conformação de Ácido Nucleico , Conformação Proteica , RNA Guia/genética
19.
Proc Natl Acad Sci U S A ; 115(20): E4604-E4612, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712837

RESUMO

RG-7916 is a first-in-class drug candidate for the treatment of spinal muscular atrophy (SMA) that functions by modulating pre-mRNA splicing of the SMN2 gene, resulting in a 2.5-fold increase in survival of motor neuron (SMN) protein level, a key protein lacking in SMA patients. RG-7916 is currently in three interventional phase 2 clinical trials for various types of SMA. In this report, we show that SMN-C2 and -C3, close analogs of RG-7916, act as selective RNA-binding ligands that modulate pre-mRNA splicing. Chemical proteomic and genomic techniques reveal that SMN-C2 directly binds to the AGGAAG motif on exon 7 of the SMN2 pre-mRNA, and promotes a conformational change in two to three unpaired nucleotides at the junction of intron 6 and exon 7 in both in vitro and in-cell models. This change creates a new functional binding surface that increases binding of the splicing modulators, far upstream element binding protein 1 (FUBP1) and its homolog, KH-type splicing regulatory protein (KHSRP), to the SMN-C2/C3-SMN2 pre-mRNA complex and enhances SMN2 splicing. These findings underscore the potential of small-molecule drugs to selectively bind RNA and modulate pre-mRNA splicing as an approach to the treatment of human disease.


Assuntos
Processamento Alternativo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Atrofia Muscular Espinal/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Transativadores/genética , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Éxons , Humanos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Conformação de Ácido Nucleico , Proteômica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/química , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Transativadores/química , Transativadores/metabolismo
20.
Nature ; 556(7701): 391-395, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29643506

RESUMO

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Domínios Proteicos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
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