RESUMEN
Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2 and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.
RESUMEN
His-Me finger (also called HNH or ßßα-me) nucleases, are a large superfamily of nucleases that share limited sequence homology, but all members carry a highly similar catalytic motif exhibiting a ßßα topology. This review represents a structural comparison of His-Me finger nucleases, summarizing their substrate-binding and recognition strategies, mechanisms of enzymatic hydrolysis, cellular functions, and the various means of activity regulation. His-Me finger nucleases usually function as monomers, making a single nick in nucleic acids to degrade foreign or host genomes, or as homodimers that introduce double-stranded DNA breaks for DNA restriction, integration, recombination, and repair. Various cellular neutralizing machineries have evolved to regulate the activity of His-Me finger nucleases, thereby maintaining genome integrity and cellular functionality.
Asunto(s)
Endonucleasas/química , Endonucleasas/metabolismo , Animales , Biocatálisis , Endonucleasas/genética , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
PKC-related serine/threonine protein kinase N1 (PKN1) is a protease/lipid-activated protein kinase that acts downstream of the RhoA and Rac1 pathways. PKN1 comprises unique regulatory, hinge region, and PKC homologous catalytic domains. The regulatory domain harbors two homologous regions, i.e., HR1 and C2-like. HR1 consists of three heptad repeats (HR1a, HR1b, and HR1c), with PKN1-(HR1a) hosting an amphipathic high-affinity cardiolipin-binding site for phospholipid interactions. Cardiolipin and C18:1 oleic acid are the most potent lipid activators of PKN1. PKN1-(C2) contains a pseudosubstrate sequence overlapping that of C20:4 arachidonic acid. However, the cardiolipin-binding site(s) within PKN1-(C2) and the respective binding properties remain unclear. Herein, we reveal (i) that the primary PKN1-(C2) sequence contains conserved amphipathic cardiolipin-binding motif(s); (ii) that trimeric PKN1-(C2) predominantly adopts a ß-stranded conformation; (iii) that two distinct types of cardiolipin (or phosphatidic acid) binding occur, with the hydrophobic component playing a key role at higher salt levels; (iv) the multiplicity of C18 fatty acid binding to PKN1-(C2); and (v) the relevance of our lipid-binding parameters for PKN1-(C2) in terms of kinetic parameters previously determined for the full-length PKN1 enzyme. Thus, our discoveries create opportunities to design specific mammalian cell inhibitors that disrupt the localization of membrane-associated PKN1 signaling molecules.
Asunto(s)
Cardiolipinas , Proteína Quinasa C , Animales , Proteína Quinasa C/metabolismo , Serina , Treonina , RatasRESUMEN
DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B-3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.
Asunto(s)
Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/química , Metilación de ADN , Dominio Catalítico , Citosina/metabolismo , ADN/química , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , ADN Metiltransferasa 3BRESUMEN
Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.
Asunto(s)
Proteínas 14-3-3/química , Biomarcadores de Tumor/química , ADN de Cadena Simple/química , Exorribonucleasas/química , Magnesio/química , Proteínas Mitocondriales/química , Oligorribonucleótidos/química , ARN/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sitios de Unión , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cationes Bivalentes , Clonación Molecular , Cristalografía por Rayos X , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Magnesio/metabolismo , Mitocondrias/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , ARN/genética , ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5' end of nascent DNA. A subsequent 5'-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5'-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA-DNA junctions to remove the 5'-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA-DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA-DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5'-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.
Asunto(s)
Replicación del ADN , ADN Mitocondrial/genética , Endonucleasas/metabolismo , Exonucleasas/metabolismo , Ribonucleasa H/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Endonucleasas/genética , Exonucleasas/genética , Humanos , Mitocondrias/genética , Nucleótidos/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , ARN/genética , Ribonucleótidos/metabolismoRESUMEN
Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple Iâ¢U and Uâ¢I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca2+-dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple Iâ¢U and Uâ¢I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca2+-binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , MicroARNs/metabolismo , Estabilidad del ARN , Ribonucleasas/metabolismo , Proteínas de Caenorhabditis elegans/química , Calcio/metabolismo , Dominio Catalítico , Inosina/análisis , ARN/metabolismo , ARN Bicatenario/química , ARN Bicatenario/metabolismo , Ribonucleasas/químicaRESUMEN
Human polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3'-to-5' exoribonuclease principally located in mitochondria where it is responsible for RNA turnover and import. Mutations in PNPase impair structured RNA transport into mitochondria, resulting in mitochondrial dysfunction and disease. PNPase is a trimeric protein with a doughnut-shaped structure hosting a central channel for single-stranded RNA binding and degradation. Here, we show that the disease-linked human PNPase mutants, Q387R and E475G, form dimers, not trimers, and have significantly lower RNA binding and degradation activities compared to wild-type trimeric PNPase. Moreover, S1 domain-truncated PNPase binds single-stranded RNA but not the stem-loop signature motif of imported structured RNA, suggesting that the S1 domain is responsible for binding structured RNAs. We further determined the crystal structure of dimeric PNPase at a resolution of 2.8 Å and, combined with small-angle X-ray scattering, show that the RNA-binding K homology and S1 domains are relatively inaccessible in the dimeric assembly. Taken together, these results show that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with destructive RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria disorders.
Asunto(s)
Mutación con Pérdida de Función , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Mutación Missense , Mutación Puntual , Polirribonucleótido Nucleotidiltransferasa/química , Estabilidad del ARN , ARN/metabolismo , Transporte Biológico , Cristalografía por Rayos X , Dimerización , Humanos , Secuencias Invertidas Repetidas , Enfermedades Mitocondriales/enzimología , Modelos Moleculares , Polirribonucleótido Nucleotidiltransferasa/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , ARN/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo PequeñoRESUMEN
RNase R is a conserved exoribonuclease in the RNase II family that primarily participates in RNA decay in all kingdoms of life. RNase R degrades duplex RNA with a 3' overhang, suggesting that it has RNA unwinding activity in addition to its 3'-to-5' exoribonuclease activity. However, how RNase R coordinates RNA binding with unwinding to degrade RNA remains elusive. Here, we report the crystal structure of a truncated form of Escherichia coli RNase R (residues 87-725) at a resolution of 1.85 Å. Structural comparisons with other RNase II family proteins reveal two open RNA-binding channels in RNase R and suggest a tri-helix 'wedge' region in the RNB domain that may induce RNA unwinding. We constructed two tri-helix wedge mutants and they indeed lost their RNA unwinding but not RNA binding or degrading activities. Our results suggest that the duplex RNA with an overhang is bound in the two RNA-binding channels in RNase R. The 3' overhang is threaded into the active site and the duplex RNA is unwound upon reaching the wedge region during RNA degradation. Thus, RNase R is a proficient enzyme, capable of concurrently binding, unwinding and degrading structured RNA in a highly processive manner during RNA decay.
Asunto(s)
Proteínas de Escherichia coli/química , Exorribonucleasas/química , Conformación de Ácido Nucleico , Dominios Proteicos , ARN Bacteriano/química , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Cinética , Modelos Moleculares , Mutación , Unión Proteica , División del ARN , Estabilidad del ARN , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ßßα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3'-to-5' exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes.
Asunto(s)
ADN/química , Endodesoxirribonucleasas/química , Secuencia de Aminoácidos , ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Activación Enzimática , Hidrólisis , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Multimerización de Proteína , Relación Estructura-ActividadRESUMEN
DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3' end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3' end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3' overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V-dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3'-end processing.
Asunto(s)
Reparación del ADN/fisiología , ADN/metabolismo , Exorribonucleasas/fisiología , Región de Flanqueo 3' , Cristalografía por Rayos X , ADN/química , Exorribonucleasas/química , Exorribonucleasas/genética , Modelos Genéticos , Conformación de Ácido NucleicoRESUMEN
In viral proteins, labile Zn-sites, where Zn(2+) is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn(2+) from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the antialcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn-ejectors.
Asunto(s)
Antivirales/farmacología , Disulfiram/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Disuasivos de Alcohol/farmacología , Línea Celular , Humanos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Compuestos de Zinc/antagonistas & inhibidores , Compuestos de Zinc/metabolismoRESUMEN
TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved ß-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.
Asunto(s)
ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ARN/química , Secuencias de Aminoácidos , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Modelos Moleculares , Mutación , Unión Proteica , ARN/química , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg(2+)-dependent 3'-5' exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H2O2-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 Å, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mutational studies further identify important conserved metal ion-binding and catalytic residues in the TatD active site for DNA hydrolysis. We thus conclude that TatD is a new class of TIM-barrel 3'-5' exonuclease that not only degrades chromosomal DNA during apoptosis but also processes single-stranded DNA during DNA repair.
Asunto(s)
Enzimas Reparadoras del ADN/química , Reparación del ADN , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Exodesoxirribonucleasas/química , Exonucleasas/química , Dominio Catalítico , ADN/química , ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasas/metabolismo , Exonucleasas/genética , Magnesio/metabolismo , Modelos Moleculares , ARN/metabolismo , Alineación de SecuenciaRESUMEN
TDP-43 is the major pathological protein identified in the cellular inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenic forms of TDP-43 are processed C-terminal fragments containing a truncated RNA-recognition motif (RRM2) and a glycine-rich region. Although extensive studies have focused on this protein, it remains unclear how the dimeric full-length TDP-43 is folded and assembled and how the processed C-terminal fragments are misfolded and aggregated. Here, using size-exclusion chromatography, pulldown assays, and small angle x-ray scattering, we show that the C-terminal-deleted TDP-43 without the glycine-rich tail is sufficient to form a head-to-head homodimer primarily via its N-terminal domain. The truncated RRM2, as well as two ß-strands within the RRM2, form fibrils in vitro with a similar amyloid-negative staining property to those of TDP-43 pathogenic fibrils in diseases. In addition to the glycine-rich region, the truncated RRM2, but not the intact RRM2, plays a key role in forming cytoplasmic inclusions in neuronal cells. Our data thus suggest that the process that disrupts the dimeric structure, such as the proteolytic cleavage of TDP-43 within the RRM2 that removes the N-terminal dimerization domain, may produce unassembled truncated RRM2 fragments with abnormally exposed ß-strands, which can oligomerize into high-order inclusions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Secuencias de Aminoácidos , Proteínas Amiloidogénicas/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Benzotiazoles , Cromatografía/métodos , Dicroismo Circular , ADN Complementario/metabolismo , Proteínas de Unión al ADN/fisiología , Dimerización , Degeneración Lobar Frontotemporal/metabolismo , Glutatión Transferasa/metabolismo , Glicina/química , Humanos , Péptidos/química , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Tiazoles/química , Rayos XRESUMEN
Exonucleases are key enzymes in the maintenance of genome stability, processing of immature RNA precursors and degradation of unnecessary nucleic acids. However, it remains unclear how exonucleases digest nucleic acids to generate correct end products for next-step processing. Here we show how the exonuclease RNase T stops its trimming precisely. The crystal structures of RNase T in complex with a stem-loop DNA, a GG dinucleotide and single-stranded DNA with different 3'-end sequences demonstrate why a duplex with a short 3'-overhang, a dinucleotide and a ssDNA with a 3'-end C cannot be further digested by RNase T. Several hydrophobic residues in RNase T change their conformation upon substrate binding and induce an active or inactive conformation in the active site that construct a precise machine to determine which substrate should be digested based on its sequence, length and structure. These studies thus provide mechanistic insights into how RNase T prevents over digestion of its various substrates, and the results can be extrapolated to the thousands of members of the DEDDh family of exonucleases.
Asunto(s)
Exorribonucleasas/química , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Exorribonucleasas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleótidos/química , Unión Proteica , Especificidad por SustratoRESUMEN
Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease that degrades specific mRNA and miRNA, and imports RNA into mitochondria, and thus regulates diverse physiological processes, including cellular senescence and homeostasis. However, the RNA-processing mechanism by hPNPase, particularly how RNA is bound via its various domains, remains obscure. Here, we report the crystal structure of an S1 domain-truncated hPNPase at a resolution of 2.1 Å. The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3' tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.
Asunto(s)
Exorribonucleasas/química , Polirribonucleótido Nucleotidiltransferasa/química , ARN/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Polirribonucleótido Nucleotidiltransferasa/genética , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Eliminación de SecuenciaRESUMEN
Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αß topology with the two ßßα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.
Asunto(s)
Apoptosis/fisiología , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/enzimología , ADN de Helmintos/química , Proteínas Mitocondriales/química , Modelos Moleculares , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografía por Rayos X , ADN de Helmintos/genética , ADN de Helmintos/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Hidrólisis , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación Missense , Relación Estructura-ActividadRESUMEN
Labile Zn fingers (Zfs) in proteins contain Zn-bound thiolates that can react with electrophilic agents, causing Zn(2+) ejection and protein unfolding. Such labile Zfs have been shown to be Cys4 or Cys3His cores whose Zn-bound Cys have no hydrogen bonds. Our aim here is to identify labile Zfs in proteins that are promising drug targets using these features. To prove the strategy used, we showed that five proteins with predicted labile Zfs reacted with Zn-ejecting agents, whereas five proteins with no or inert Zfs did not. The comprehensive set of labile Zfs provides new drug targets and guidelines to redesign Zn-ejecting compounds with improved specificity.
Asunto(s)
Proteínas/química , Dedos de Zinc , Zinc/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Proteínas Potenciadoras de Unión a CCAAT/química , ADN (Citosina-5-)-Metiltransferasas/química , Disulfuros/química , Histona Desacetilasas/química , Humanos , Proteínas Recombinantes/química , Compuestos de Sulfhidrilo/química , Factor 6 Asociado a Receptor de TNF/química , Tiram/química , Ubiquitina-Proteína Ligasas/químicaRESUMEN
RNA maturation relies on various exonucleases to remove nucleotides successively from the 5' or 3' end of nucleic acids. However, little is known regarding the molecular basis for substrate and cleavage preference of exonucleases. Our biochemical and structural analyses on RNase T-DNA complexes show that the RNase T dimer has an ideal architecture for binding a duplex with a short 3' overhang to produce a digestion product of a duplex with a 2-nucleotide (nt) or 1-nt 3' overhang, depending on the composition of the last base pair in the duplex. A 'C-filter' in RNase T screens out the nucleic acids with 3'-terminal cytosines for hydrolysis by inducing a disruptive conformational change at the active site. Our results reveal the general principles and the working mechanism for the final trimming step made by RNase T in the maturation of stable RNA and pave the way for the understanding of other DEDD family exonucleases.