RESUMEN
DNA transposition plays key roles in genome diversity, pathogenesis, and evolution. Yet, structural and mechanistic information on transposition targeting and regulation is limited. Arias-Palomo and Berger now define the decameric organization of the AAA+ ATPase IstB, unveiling key insights into its targeting and regulation of IstA transposase activity.
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Bacterias/genética , Bacterias/metabolismo , Elementos Transponibles de ADNRESUMEN
Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Replicación del ADN/genética , ADN/genética , Exonucleasas/genética , Inestabilidad Genómica/genética , RecQ Helicasas/genética , Línea Celular , Línea Celular Tumoral , Daño del ADN/genética , ADN Helicasas/genética , Análisis Mutacional de ADN/métodos , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Humanos , Mutación/genética , Oncogenes/genética , Fosforilación/genética , Regulación hacia Arriba/genéticaRESUMEN
TET-mediated 5-methyl cytosine (5mC) oxidation acts in epigenetic regulation, stem cell development, and cancer. Hu et al. now determine the crystal structure of the TET2 catalytic domain bound to DNA, shedding light on 5mC-DNA substrate recognition and the catalytic mechanism of 5mC oxidation.
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5-Metilcitosina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Dioxigenasas , HumanosRESUMEN
Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.
Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.
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Reparación del ADN , Factor de Transcripción TFIIH , ADN/metabolismo , Daño del ADN , ADN de Cadena Simple , Endonucleasas/metabolismo , Nucleótidos , Factor de Transcripción TFIIH/metabolismoRESUMEN
The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.
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Reparación del ADN , Proteína de Replicación A , Proteína de la Xerodermia Pigmentosa del Grupo A , ADN/metabolismo , Daño del ADN , Unión Proteica , Dominios Proteicos , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.
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Cobre/metabolismo , Glicina/análogos & derivados , Oxígeno/metabolismo , Catálisis , Dominio Catalítico/fisiología , Cristalografía por Rayos X/métodos , Cisteína/metabolismo , Glicina/metabolismo , Oxidación-Reducción , Sulfatasas/metabolismoRESUMEN
Histone lysine methylation and demethylation regulate histone methylation dynamics, which impacts chromatin structure and function. To read and erase the methylated histone residues, lysine demethylases must specifically recognize the histone sequences and methylated sites and discriminate the degree of these methylations. In this issue of Genes & Development, Cheng and colleagues (pp. 1758-1771) determine a crystal structure of histone lysine demethylase KDM2A that specifically targets lower degrees of H3K36 methylation. The results reveal the structural basis for H3K36 substrate specificity and suggest mechanisms of Lys36 demethylation. This KDM2A-H3K36 complex structure, coupled with functional studies, provides needed insight into the process and regulation of histone demethylation.
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Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Modelos Moleculares , AnimalesRESUMEN
Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 µm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.
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Proteínas Bacterianas/química , ADN Helicasas/química , Oxidorreductasas/química , Percloratos/química , Rhodocyclaceae/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , ADN Helicasas/genética , ADN Helicasas/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Percloratos/metabolismo , Rhodocyclaceae/genéticaRESUMEN
Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life - the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
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Reparación del ADN , Replicación del ADN , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Animales , ADN/química , ADN/genética , ADN/metabolismo , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging.
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Reparación de la Incompatibilidad de ADN , Oro/química , Nanopartículas/química , Conformación de Ácido Nucleico , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Algoritmos , ADN Bacteriano/química , ADN Bacteriano/metabolismo , ADN Bacteriano/ultraestructura , Proteínas de Escherichia coli/metabolismo , Cinética , Microscopía Electrónica de Transmisión , Modelos Moleculares , Proteínas MutL , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Nanopartículas/ultraestructura , Unión Proteica , SolucionesRESUMEN
Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688-698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Salmonella typhimurium/metabolismo , Factor sigma/química , Factor sigma/metabolismoRESUMEN
Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.
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Proteínas de Unión a Hierro/química , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Azufre/química , Cisteína/química , Humanos , Proteínas de Unión a Hierro/fisiología , Proteínas Hierro-Azufre/biosíntesis , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/fisiología , Unión Proteica/fisiología , Especificidad por Sustrato/fisiología , Azufre/metabolismo , FrataxinaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismoRESUMEN
BACKGROUND: Wearable exoskeletons are a recently developed technology. OBJECTIVES: The present systematic review aimed to investigate the effect of a wearable exoskeleton on post-stroke walking by considering its use in a gait training system and simply as an orthosis assisting walking. METHODS: We systematically searched for randomised and quasi-randomised controlled trials in PubMed, Scopus, CINAHL and Embase databases from their earliest publication record to July 2021. We chose reports of trials investigating the effects of exoskeleton-assisted training or the effects of wearing an exoskeleton to assist walking. A meta-analysis was conducted to explore the benefits of the wearable exoskeleton on mobility capacity, walking speed, motor function, balance, endurance and activities of daily living. RESULTS: We included 13 studies (492 participants) comparing exoskeleton-assisted training with dose-matched conventional gait training. Studies addressing the effect of wearing a wearable exoskeleton were unavailable. As compared with conventional gait training at the end of the intervention, exoskeleton-assisted training was superior for walking speed (mean difference [MD] 0.13 m/s, 95% CI 0.05; 0.21) and balance (standardized MD [SMD] 0.3, 95% CI 0.07; 0.54). The subgroup with chronic stroke (i.e., > 6 months) presented the outcome favouring exoskeleton-assisted training regarding overall mobility capacity (SMD 0.37, 95% CI 0.04; 0.69). At the end of follow-up, exoskeleton-assisted training was superior to conventional gait training in overall mobility (SMD 0.45, 95% CI 0.07; 0.84) and endurance (MD 46.23 m, 95% CI 9.90; 82.56). CONCLUSIONS: Exoskeleton-assisted training was superior to dose-matched conventional gait training in several gait-related outcomes at the end of the intervention and follow-up in this systematic review and meta-analysis, which may support the use of exoskeleton-assisted training in the rehabilitation setting. Whether wearing versus not wearing a wearable exoskeleton is beneficial during walking remains unknown.
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Dispositivo Exoesqueleto , Rehabilitación de Accidente Cerebrovascular , Accidente Cerebrovascular , Dispositivos Electrónicos Vestibles , Humanos , Actividades Cotidianas , Marcha , CaminataRESUMEN
Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.
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ADN Helicasas , Reparación del ADN , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Conformación Molecular , ADN/metabolismo , Transcripción GenéticaRESUMEN
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the â¼15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn â¼ I154F > W155F > W155A â¼ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.
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Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Regulación Alostérica , Liasas de Carbono-Azufre/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Sulfuros/metabolismo , FrataxinaRESUMEN
All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.
RESUMEN
BACKGROUND: Trials that assessed the impact of protein supplementation on endurance training adaptations have reported conflicting findings. OBJECTIVE: To determine the impact of protein supplementation during chronic endurance training on aerobic capacity, body composition and exercise performance in healthy and clinical populations. DESIGN: A systematic database search was conducted for randomised controlled trials addressing the effects of protein supplementation during endurance training on aerobic capacity, body composition and exercise performance in PubMed, Embase, Web of Science, and CINAHL. Meta-analyses were performed to outline the overall effects of protein supplementation with all studies containing endurance training components. The effects of endurance training and add-on effects of protein supplementation were evaluated by the meta-analyses with endurance training-focused studies. RESULTS: Nineteen studies and 1162 participants contributed to the analyses. Compared with the control group, the protein supplementation group demonstrated greater improvements in aerobic capacity measured by mixed peak oxygen uptake (VÌO2peak) and peak workload power (Wpeak) (standardised mean difference [SMD] = 0.36, 95% confidence interval [CI]: 0.05 to 0.67), and VÌO2peak (mean difference [MD] = 0.89 mLâ§kg-1â§min-1, 95% CI: 0.07 to 1.70); had a greater lean mass gain (MD = 0.32 kg, 95% CI: 0.07 to 0.58); and had a greater improvement in time trial performance (MD = -29.1s, 95% CI:-55.3 to -3.0). Secondary analyses showed that, in addition to the substantial improvement in VÌO2peak (MD = 3.67 mLâ§kg-1â§min-1, 95% CI: 2.32 to 5.03) attributed to endurance training, protein supplementation provided an additional 26.4% gain in VÌO2peak (MD = 0.97 mLâ§kg-1â§min-1, 95% CI: -0.03 to 1.97). CONCLUSION: Protein supplementation further increased aerobic capacity, stimulated lean mass gain, and improved time trial performance during chronic endurance training in healthy and clinical populations. PROSPERO REGISTRATION NUMBER: (CRD42020155239).
Asunto(s)
Proteínas en la Dieta/farmacología , Suplementos Dietéticos , Entrenamiento Aeróbico/métodos , Resistencia Física/efectos de los fármacos , Adaptación Fisiológica , Composición Corporal/efectos de los fármacos , Proteínas en la Dieta/administración & dosificación , HumanosRESUMEN
DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.
RESUMEN
The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates Pol II to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in vitro in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.