RESUMEN
Nucleotide excision repair removes DNA lesions caused by ultraviolet light, cisplatin-like compounds and bulky adducts1. After initial recognition by XPC in global genome repair or a stalled RNA polymerase in transcription-coupled repair, damaged DNA is transferred to the seven-subunit TFIIH core complex (Core7) for verification and dual incisions by the XPF and XPG nucleases2. Structures capturing lesion recognition by the yeast XPC homologue Rad4 and TFIIH in transcription initiation or DNA repair have been separately reported3-7. How two different lesion recognition pathways converge and how the XPB and XPD helicases of Core7 move the DNA lesion for verification are unclear. Here we report on structures revealing DNA lesion recognition by human XPC and DNA lesion hand-off from XPC to Core7 and XPA. XPA, which binds between XPB and XPD, kinks the DNA duplex and shifts XPC and the DNA lesion by nearly a helical turn relative to Core7. The DNA lesion is thus positioned outside of Core7, as would occur with RNA polymerase. XPB and XPD, which track the lesion-containing strand but translocate DNA in opposite directions, push and pull the lesion-containing strand into XPD for verification.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , ADN , Factor de Transcripción TFIIH , Proteína de la Xerodermia Pigmentosa del Grupo A , Humanos , ADN/química , ADN/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción TFIIH/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Especificidad por Sustrato , ARN Polimerasas Dirigidas por ADN/metabolismoRESUMEN
The study was aimed to validate the efficacy of the pulsed Nd:YAG laser on nerve regeneration in a rat sciatic nerve crushed model. 54 Wistar rats were randomly assigned into three groups: shame control, crush control, and laser treated group. For the laser treated group, the pulsed Nd:YAG laser (10 Hz) with 350 mJ per pulse in energy density and 50 J/cm2 in fluence was applied extracorporeally at the lesion site for 12 min to daily deliver 500 J immediately and consecutive 9 days following the crush injury. At week 1, the apoptosis-related activities in the injured nerve were examined (n = 8/each group). The sciatic functional index (SFI) was measured preoperatively and weekly until 4 weeks after the index procedure. The injured nerve and the innervated gastrocnemius muscle histology were assessed at week 4 (n = 10/each group). At week 1, the laser group showed the significant less TUNEL-positive ratio (P < 0.05), and the lower expression of cleaved caspase3/procaspase-3 and beclin-2/beclin-2-associated protein X ratios compared with the crush control. Furthermore, the laser group revealed significantly better SFI since week 1 and throughout the study (P < 0.05, all) compared with the crush control. At week 4, the laser group showed significantly higher axon density, lower myelin g-ratio, and the corresponding higher glycogen expression (P < 0.05, all) in the gastrocnemius muscle compared with those in the crush control. The pulsed Nd:YAG might enhance the injured nerve regeneration via apoptosis inhibition.
Asunto(s)
Lesiones por Aplastamiento , Terapia por Láser , Láseres de Estado Sólido , Neuropatía Ciática , Ratas , Animales , Ratas Wistar , Compresión Nerviosa , Nervio Ciático/lesiones , Regeneración Nerviosa/fisiología , Neuropatía Ciática/patologíaRESUMEN
Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.
Asunto(s)
Aductos de ADN/genética , Proteínas de Unión al ADN/fisiología , Factor de Transcripción TFIIH/fisiología , Proteína de la Xerodermia Pigmentosa del Grupo A/fisiología , Animales , Cisplatino/química , Aductos de ADN/química , Reparación del ADN , ADN de Cadena Simple/fisiología , Proteínas de Unión al ADN/química , Ensayo de Cambio de Movilidad Electroforética , Humanos , Unión Proteica , Células Sf9 , Spodoptera , Factor de Transcripción TFIIH/química , Proteína de la Xerodermia Pigmentosa del Grupo A/químicaRESUMEN
Apoptosis has emerged as a primary cause of tendinopathy. CD44 signaling pathways exert anti-apoptotic and -inflammatory effects on tumor cells, chondrocytes, and fibroblast-like synoviocytes. The aim of this study was to examine the association among CD44, apoptosis, and inflammation in tendinopathy. Expression of CD44 and apoptotic cell numbers in tendon tissue from patients with long head of biceps (LHB) tendinopathy were determined according to the histological grades of tendinopathy. Primary tenocytes from Achilles tendon of Sprague-Dawley rats 1 week after collagenase injection were cultured with an antagonizing antibody against CD44. Treatment responses were determined by evaluating cell viability and expression of tendon-related proliferation markers, inflammatory mediators, and apoptosis. The expression of CD44 and apoptosis were positively correlated with the severity of tendinopathy in the human LHB tendinopathy. Furthermore, CD44 expression and apoptotic cells were co-stained in tendinopathic tendon. Blocking the CD44 signaling pathways in rat primary tenocytes by OX-50 induced cell apoptosis and the elevated levels of cleaved caspase-3. Furthermore, they had decreased cell viability and expression of collagen type I, type III, tenomodulin, and phosphorylated AKT. In contrast, there were elevated levels of inflammatory mediators, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, cyclooxygenase-2, and phosphorylated NF-κB, as well as matrix metalloproteinase (MMP) family members including MMP-1, -3, -9, and -13 in tenocytes upon OX-50 treatment. This study is the first to demonstrate the association of CD44 and apoptosis in tendinopathy. Our data imply that CD44 may play a role in tendinopathy via regulating apoptosis, inflammation, and extracellular matrix homeostasis.
Asunto(s)
Apoptosis , Receptores de Hialuranos/metabolismo , Mediadores de Inflamación/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Masculino , Metaloproteinasas de la Matriz/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Dióxido de Silicio/toxicidad , Tendinopatía/metabolismo , Tendinopatía/patología , Tenocitos/citología , Tenocitos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/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.
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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
BACKGROUND: Fat embolism syndrome (FES) is a rare complication that can occur between 12 and 72 h after the initial insult. Isolated cerebral FES without pulmonary symptoms is rarer. Early fracture fixation might prevent FES. We report a case of multiple-fracture with FES despite definite fixation three hours post-injury. CASE PRESENTATION: A 54-year-old man presented with multiple fractures: left femoral shaft (AO B2), left distal radius (AO C3), left comminuted patella, right comminuted 1st metatarsal base and left 2nd-4th metatarsal neck. Because he was stable, we gave him early total care and definite fixation, which required seven hours and yielded no complications. After he recovered from anesthesia, however, his eyes deviated right, his right upper arm was paralyzed, his consciousness level was poor, and his Glasgow Coma Scale score was E3VeM4. Chest X-rays showed clear lung fields, and brain computed tomography showed no intracranial hemorrhage. He did, however, have tachycardia, anemia, and thrombocytopenia. Brain magnetic resonance images showed a hyperintensive starfield pattern on diffuse weighted images, which suggested cerebral FES. After supportive care, his consciousness cleared on postoperative day 17, and he recovered full right upper arm muscle power after four months; however, he had a significant cognitive deficit. One-year post-injury, after regular rehabilitation therapy, he was able to independently perform his activities of daily living but still had a residual mild cognitive deficit. CONCLUSION: Early fixation can attenuate but not eliminate the incidence of FES. Early assessment and rehabilitation therapy might be required for patients with cerebral FES and cognitive deficits; however, such deficits are difficult to predict and need long-term follow-ups.
Asunto(s)
Embolia Grasa/diagnóstico , Embolia Intracraneal/diagnóstico , Traumatismo Múltiple/complicaciones , Actividades Cotidianas , Encéfalo/diagnóstico por imagen , Embolia Grasa/etiología , Embolia Grasa/rehabilitación , Fijación de Fractura , Humanos , Embolia Intracraneal/etiología , Embolia Intracraneal/rehabilitación , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/cirugía , Factores de Tiempo , Resultado del TratamientoRESUMEN
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
Background: to compare the 52-week effectiveness and safety between HYAJOINT Plus (HJP) and Durolane in knee osteoarthritis (OA) treatment. Methods: consecutive patients received a single injection of 3 mL HJP or Durolane. The primary outcome was a visual analog scale (VAS) pain measurement at 26 weeks post-injection. Secondary outcomes included other clinical, satisfaction, and safety assessments for 52 weeks. Results: 142 patients were equally randomized. At week 26, the HJP group had less VAS pain than the Durolane group (18.1 ± 9.5 versus 24.4 ± 14.0, p = 0.001). Both groups showed improvement in their VAS pain and stiffness scores, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and total scores for 52 weeks after injection (p < 0.001). However, the HJP group showed lower VAS pain and stiffness scores, reduced WOMAC pain and stiffness scores, a shorter Timed "Up & Go" (TUG) time, and a higher satisfaction score than the Durolane group for 39 weeks (p < 0.05). Only mild and self-limited adverse events occurred (40.8%). Conclusion: While a single injection of either HJP or Durolane is safe and effective for at least 52 weeks, HJP provided superior improvement in terms of VAS pain and stiffness scores, WOMAC pain and stiffness scores, and satisfaction score within 39 weeks of treatment.
RESUMEN
Aeromonas hydrophilia can cause soft tissue infection in both immunocompromised and healthy persons. A healthy 15-year-old adolescent fell into a ditch after a scooter accident and sustained a right distal tibial shaft closed fracture, a right femoral shaft closed fracture, and a dirty laceration over the medial aspect of the distal thigh above the right knee. After empiric antibiotics and radical debridement of the contaminated wound, a femoral interlocking nail and tibial external fixator were applied. However, acute osteomyelitis later presented in his femur and tibia, and Aeromonas hydrophilia grew in cultures from the knee wound and the fracture sites. During the follow-up, his tibia became an infected nonunion, and was successfully treated with the induced membrane technique. In an otherwise healthy patient with a closed fracture, Aeromonas hydrophilia can cause acute osteomyelitis and necrotizing fasciitis by spreading from a nearby contaminated wound. Exposure to water is a risk factor for Aeromonas hydrophilia infection.
Asunto(s)
Aeromonas , Fracturas Cerradas , Fracturas de la Tibia , Adolescente , Clavos Ortopédicos , Fijadores Externos , HumanosRESUMEN
Human Tudor-SN is involved in the degradation of hyper-edited inosine-containing microRNA precursors, thus linking the pathways of RNA interference and editing. Tudor-SN contains four tandem repeats of staphylococcal nuclease-like domains (SN1-SN4) followed by a tudor and C-terminal SN domain (SN5). Here, we showed that Tudor-SN requires tandem repeats of SN domains for its RNA binding and cleavage activity. The crystal structure of a 64-kD truncated form of human Tudor-SN further shows that the four domains, SN3, SN4, tudor and SN5, assemble into a crescent-shaped structure. A concave basic surface formed jointly by SN3 and SN4 domains is likely involved in RNA binding, where citrate ions are bound at the putative RNase active sites. Additional modeling studies provide a structural basis for Tudor-SN's preference in cleaving RNA containing multiple I.U wobble-paired sequences. Collectively, these results suggest that tandem repeats of SN domains in Tudor-SN function as a clamp to capture RNA substrates.
Asunto(s)
Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Cristalografía por Rayos X , Endonucleasas , Humanos , Nucleasa Microcócica/química , Modelos Moleculares , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , ARN/metabolismo , Edición de ARN , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Secuencias Repetitivas de AminoácidoRESUMEN
Nonspecific endonucleases hydrolyze DNA without sequence specificity but with sequence preference, however the structural basis for cleavage preference remains elusive. We show here that the nonspecific endonuclease ColE7 cleaves DNA with a preference for making nicks after (at 3'O-side) thymine bases but the periplasmic nuclease Vvn cleaves DNA more evenly with little sequence preference. The crystal structure of the 'preferred complex' of the nuclease domain of ColE7 bound to an 18 bp DNA with a thymine before the scissile phosphate had a more distorted DNA phosphate backbone than the backbones in the non-preferred complexes, so that the scissile phosphate was compositionally closer to the endonuclease active site resulting in more efficient DNA cleavage. On the other hand, in the crystal structure of Vvn in complex with a 16 bp DNA, the DNA phosphate backbone was similar and not distorted in comparison with that of a previously reported complex of Vvn with a different DNA sequence. Taken together these results suggest a general structural basis for the sequence-dependent DNA cleavage catalyzed by nonspecific endonucleases, indicating that nonspecific nucleases could induce DNA to deform to distinctive levels depending on the local sequence leading to different cleavage rates along the DNA chain.
Asunto(s)
Proteínas Bacterianas/química , Colicinas/química , ADN/química , Desoxirribonucleasas/química , Endonucleasas/química , Proteínas de Escherichia coli/química , Esterasas/química , Modelos Moleculares , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Colicinas/genética , Colicinas/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Esterasas/genética , Esterasas/metabolismo , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Purpose: The purpose of the current study was to evaluate the effectiveness of ultrasound-guided percutaneous carpal tunnel release in hemodialysis patients with carpal tunnel syndrome. Methods: From February 2009 to April 2013, a prospective review of 113 consecutive cases of ultrasound-guided percutaneous carpal tunnel release was carried out in 84 hemodialysis patients. Results were analyzed by clinical subjective scale, two self-administered questionnaires, and functional evaluations at seven time points (1 week and 1, 3, 6, 12, 18, and 24 months). Results: Satisfactory symptom improvement in patients was 82%, 80%, 86%, 89%, 90%, 91%, and 90% at 1 week and 1, 3, 6, 12, 18, and 24 months postoperatively, respectively. Moderate pain was suffered in 11.5% of patients within 1 week, 8.8% within 1 month, 2.7% within 3 months, and none after 12 months postoperatively. Static two-point discrimination and Semmes-Weinstein monofilament examinations presented significant improvements after 1 week and 1 month postoperatively and with time. Postoperative grip power demonstrated recovery and a significant increase after 3 and 6 months postoperatively. Three-jaw chuck-pinch strength showed significant increase after 1 month postoperatively. There were no operative complications. Conclusion: Ultrasound-guided percutaneous carpal tunnel release is an effective and safe procedure in hemodialysis patients with carpal tunnel syndrome. The advantages include a less invasive procedure, no tourniquet needed, only limited infiltration anesthesia, minimal soft-tissue exploration, and relatively short operation time. Our data suggest this technique can reliably relieve clinical symptoms, with early restoration of grip and pinch strength.
RESUMEN
PURPOSE: To mitigate the risk of poor wound healing and of infection associated with the open repair of Achilles tendon midsubstance ruptures, minimally invasive techniques have been developed. We report our preliminary results after reviewing our "jigless knotless internal brace technique." METHODS: Patients were placed in prone position and a transverse 3-cm incision was made proximal to the palpable ruptured end. The proximal ruptured end was pulled out, gently debrided, and sutured using Krackow locking loops. Percutaneous sutures were crisscrossed through the distal tendon stump and looped around the Krackow sutures over the proximal stump. The ipsilateral Krackow sutures and the contralateral crisscrossed sutures were subcutaneously passed through two mini-incisions over the posterior calcaneus tuberosity and seated at the tuberosity with two 4.5-mm knotless suture anchors. All patients underwent the same post-operative rehabilitation protocol and regular follow-ups for at least 1 year. RESULTS: We recruited 10 patients (mean age, 37.3 years) who scored 100 points on the American Orthopaedic Foot and Ankle Society (AOFAS) scale, and who returned to their preoperative exercise levels 1-year post-operatively with no complications. CONCLUSION: Our method is simple, effective, and requires no special tools. It might be a reliable option for Achilles tendon repair. LEVEL OF EVIDENCE: III.
Asunto(s)
Tendón Calcáneo/lesiones , Tendón Calcáneo/cirugía , Desbridamiento/métodos , Rotura/cirugía , Técnicas de Sutura , Traumatismos de los Tendones/cirugía , Adulto , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Rotura/diagnóstico , Anclas para Sutura , Traumatismos de los Tendones/diagnóstico , Adulto JovenRESUMEN
In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way.
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Daño del ADN , Reparación del ADN , ADN/química , Desoxirribonucleasas/química , Modelos Químicos , Modelos Moleculares , ARN/química , Ribonucleasas/química , Endonucleasas/química , Activación EnzimáticaRESUMEN
The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.
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Proteínas Bacterianas/química , Colicinas/química , Proteínas de Escherichia coli/química , Níquel/química , Zinc/química , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cationes Bivalentes/metabolismo , Colicinas/genética , Colicinas/metabolismo , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Estabilidad de Enzimas , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Histidina/química , Histidina/genética , Modelos Moleculares , Mutación/genética , Níquel/farmacología , Unión Proteica , Conformación Proteica , Zinc/farmacologíaRESUMEN
RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3'-to-5' exonuclease activity is blocked by a 3'-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3'-terminal nucleobases. To elucidate if the π-π stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein-nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specificity with wild-type RNase T. However, the Y mutants exhibited altered sequence-dependent activity, digesting ssDNA with both 3'-end CC and GG sequences. Moreover, the W and Y mutants had reduced DNA-binding activity and lower thermal stability as compared to wild-type RNase T. Taken together, our results suggest that the four phenylalanine residues in RNase T not only play critical roles in sequence-specific recognition, but also in overall protein stability. Our results provide the first evidence showing that the π-π stacking interactions between nucleobases and protein aromatic residues may guide the sequence-specific activity for DNA and RNA enzymes.
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ADN de Cadena Simple/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/crecimiento & desarrollo , Exorribonucleasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Dicroismo Circular , Estabilidad de Enzimas , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , TemperaturaRESUMEN
Human cytosolic thymidine kinase (TK1) is tightly regulated in the cell cycle by multiple mechanisms. Our laboratory has previously shown that in mitotic-arrested cells human TK1 is phosphorylated at serine-13, accompanied by a decrease in catalytic efficiency. In this study we investigated whether serine-13 phosphorylation regulated TK1 activity and found that substitution of serine-13 with aspartic acid (S13D), which mimics phosphorylation, not only diminished the ATP-activating effect on the enzyme, but also decreased its thymidine substrate affinity. Our experimental results further showed that the S13D mutation perturbed ATP-induced tetramerization of TK1. Given that the dimeric form of TK1 is less active than the tetrameric, we propose that mitotic phosphorylation of serine-13 is of physiological importance, in that it may counteract ATP-dependent activation of TK1 by affecting its quaternary structure, thus attenuating its enzymatic function at the G2/M phase.
Asunto(s)
Adenosina Trifosfato/química , Citosol/enzimología , Mitosis , Serina/química , Timidina Quinasa/química , Adenosina Trifosfato/farmacología , Ácido Aspártico/química , Catálisis , Ciclo Celular , Línea Celular , Cromatografía , Cromatografía en Gel , Dimerización , Relación Dosis-Respuesta a Droga , Fase G2 , Humanos , Cinética , Modelos Biológicos , Mutación , Fosforilación , Plásmidos/metabolismo , Estructura Cuaternaria de Proteína , Timidina/farmacologíaRESUMEN
The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA. The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 A resolution. Vvn has a novel mixed alpha/beta topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases; however, a known 'betabetaalpha-metal' motif is identified in the central cleft region. The crystal structure of the mutant Vvn-DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by approximately 20 degrees. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme-substrate and enzyme-product structures observed in the mutant Vvn-DNA crystals, a catalytic mechanism is proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.