RESUMO
Fen1 and Mus81-Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81-Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21-26 (aa from 21-26) and N108-114 (aa from 108-114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/química , Endonucleases/química , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
MUS81 shares a high-degree homology with the catalytic XPF subunit of the XPF-ERCC1 endonuclease complex. It is catalytically active only when complexed with the regulatory subunits Mms4 or Eme1 in budding and fission yeasts, respectively, and EME1 or EME2 in humans. Although Mus81 complexes are implicated in the resolution of recombination intermediates in vivo, recombinant yeast Mus81-Mms4 and human MUS81-EME1 isolated from Escherichia coli fail to cleave intact Holliday junctions (HJs) in vitro. In this study, we show that human recombinant MUS81-EME2 isolated from E. coli cleaves HJs relatively efficiently, compared to MUS81-EME1. Furthermore, MUS81-EME2 catalyzed cleavage of nicked and gapped duplex deoxyribonucleic acids (DNAs), generating double-strand breaks. The presence of a 5' phosphate terminus at nicks and gaps rendered DNA significantly less susceptible to the cleavage by MUS81-EME2 than its absence, raising the possibility that this activity could play a role in channeling damaged DNA duplexes that are not readily repaired into the recombinational repair pathways. Significant differences in substrate specificity observed with unmodified forms of MUS81-EME1 and MUS81-EME2 suggest that they play related but non-overlapping roles in DNA transactions.
Assuntos
DNA Cruciforme/química , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/química , Endonucleases/química , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Clivagem do DNA , Escherichia coli , Humanos , Cinética , Complexos Multiproteicos/química , Subunidades Proteicas/química , Especificidade por SubstratoRESUMO
The correct removal of 5'-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Recombinação Homóloga , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Immunoblotting , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Multi-UTR genes are widely transcribed and express their alternative 3'UTR isoforms in a cell type-specific manner. As transcriptional enhancers regulate mRNA expression, we investigated if they also regulate 3'UTR isoform expression. Endogenous enhancer deletion of the multi-UTR gene PTEN did not impair transcript production but prevented 3'UTR isoform switching which was recapitulated by silencing of an enhancer-bound transcription factor. In reporter assays, enhancers increase transcript production when paired with single-UTR gene promoters. However, when combined with multi-UTR gene promoters, they change 3'UTR isoform expression by increasing 3' end processing activity of polyadenylation sites. Processing activity of polyadenylation sites is affected by transcription factors, including NF-κB and MYC, transcription elongation factors, chromatin remodelers, and histone acetyltransferases. As endogenous cell type-specific enhancers are associated with genes that increase their short 3'UTRs in a cell type-specific manner, our data suggest that transcriptional enhancers integrate cellular signals to regulate cell type-and condition-specific 3'UTR isoform expression.
Assuntos
Regulação da Expressão Gênica , Poliadenilação , Regiões 3' não Traduzidas/genética , Isoformas de Proteínas/genética , Sequências Reguladoras de Ácido NucleicoRESUMO
Wnt/ Wingless (Wg) is essential for embryonic development and adult homeostasis in all metazoans, but the mechanisms by which secreted Wnt/Wg is processed remain largely unknown. A Drosophila Sol narae (Sona) is a member of A Disintegrin And Metalloprotease with ThromboSpondin motif (ADAMTS) family, and positively regulates Wg signaling by promoting Wg secretion. Here we report that Sona and Wg are secreted by both conventional Golgi and exosomal transports, and Sona cleaves extracellular Wg at the two specific sites, leading to the generation of N-terminal domain (NTD) and C-terminal domain (CTD) fragments. The cleaved forms of extracellular Wg were detected in the extracellular region of fly wing discs, and its level was substantially reduced in sona mutants. Transient overexpression of Wg-CTD increased wing size while prolonged overexpression caused lethality and developmental defects. In contrast, Wg-NTD did not induce any phenotype. Moreover, the wing defects and lethality induced by sona RNAi were considerably rescued by Wg-CTD, indicating that a main function of extracellular Sona is the generation of Wg-CTD. Wg-CTD stabilized cytoplasmic Armadillo (Arm) and had genetic interactions with components of canonical Wg signaling. Wg-CTD also induced Wg downstream targets such as Distal-less (Dll) and Vestigial (Vg). Most importantly, Cyclin D (Cyc D) was induced by Wg-CTD but not by full-length Wg. Because Sona also induces Cyc D in a cell non-autonomous manner, Wg-CTD generated by Sona in the extracellular region activates a subset of Wg signaling whose major function is the regulation of cell proliferation.
Assuntos
Proteínas ADAMTS/metabolismo , Proliferação de Células/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Domínios Proteicos/genética , Proteína Wnt1/química , Proteína Wnt1/metabolismo , Proteínas ADAMTS/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Ciclina D/metabolismo , Proteínas de Drosophila/genética , Exossomos/metabolismo , Complexo de Golgi/metabolismo , Fenótipo , Estabilidade Proteica , Interferência de RNA , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Proteína Wnt1/genéticaRESUMO
Two-dimensional (2D) nanomaterials, such as graphene-based materials and transition metal dichalcogenide (TMD) nanosheets, are promising materials for biomedical applications owing to their remarkable cytocompatibility and physicochemical properties. On the basis of their potent antibacterial properties, 2D materials have potential as antibacterial films, wherein the 2D nanosheets are immobilized on the surface and the bacteria may contact with the basal planes of 2D nanosheets dominantly rather than contact with the sharp edges of nanosheets. To address these points, in this study, we prepared an effective antibacterial surface consisting of representative 2D materials, i.e., graphene oxide (GO) and molybdenum disulfide (MoS2), formed into nanosheets on a transparent substrate for real device applications. The antimicrobial properties of the GO-MoS2 nanocomposite surface toward the Gram-negative bacteria Escherichia coli were investigated, and the GO-MoS2 nanocomposite exhibited enhanced antimicrobial effects with increased glutathione oxidation capacity and partial conductivity. Furthermore, direct imaging of continuous morphological destruction in the individual bacterial cells having contacts with the GO-MoS2 nanocomposite surface was characterized by holotomographic (HT) microscopy, which could be used to detect the refractive index (RI) distribution of each voxel in bacterial cell and reconstruct the three-dimensional (3D) mapping images of bacteria. In this regard, the decreases in both the volume (67.2%) and the dry mass (78.8%) of bacterial cells that came in contact with the surface for 80 min were quantitatively measured, and releasing of intracellular components mediated by membrane and oxidative stress was observed. Our findings provided new insights into the antibacterial properties of 2D nanocomposite film with label-free tracing of bacterial cell which improve our understanding of antimicrobial activities and opened a window for the 2D nanocomposite as a practical antibacterial film in biomedical applications.
RESUMO
Highly conserved eukaryotic histones are polybasic proteins that package DNA into nucleosomes, a building block of chromatin, allowing extremely long DNA molecules to form compact and discrete chromosomes. The histone N-terminal tails that extend from the nucleosome core act as docking sites for many proteins through diverse post-translational modifications, regulating various DNA transactions. In this report, we present evidence that the nucleosomes can positively regulate the enzymatic activity of Rad27 (yeast Fen1), a major processing enzyme important for Okazaki fragment in eukaryotes. We found that individual histones, histone octamers, and nucleosomes are able to stimulate Rad27 in a manner dependent on the N-terminal tails of histones. Kinetic analyses suggest that an increase in catalytic efficiency of Rad27 was mainly due to increased affinity between DNA substrates and Rad27. It appears that the physical interaction in vivo between histones and Rad27 results in the enrichment of Rad27 in the vicinity of chromatin, increasing the availability of Rad27 for various DNA metabolisms. These results indicate that nucleosomes are not a mere structural component of chromatin, but an active regulator of DNA metabolisms that serves to ensure the efficient and faithful processing of structural intermediates arising during DNA transactions.