RESUMO
Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.
RESUMO
Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein-protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.
Assuntos
Monofosfato de Adenosina/metabolismo , Coenzima A Ligases/química , Modelos Moleculares , Monofosfato de Adenosina/química , Domínio Catalítico , Coenzima A Ligases/metabolismo , Cristalização , Ligantes , Estrutura Molecular , Especificidade por SubstratoRESUMO
UNLABELLED: N-acetyl- and N-glycolylneuraminic acids (Sia) and α2ß1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2ß1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2ß1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCE: Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.
Assuntos
Gangliosídeos/metabolismo , Integrina alfa2beta1/metabolismo , Ácidos Neuramínicos/metabolismo , Receptores Virais/metabolismo , Infecções por Rotavirus/metabolismo , Rotavirus/fisiologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Humanos , Integrina alfa2beta1/genética , Ácido N-Acetilneuramínico/metabolismo , Ligação Proteica , Receptores Virais/genética , Rotavirus/genética , Infecções por Rotavirus/genética , Infecções por Rotavirus/virologiaRESUMO
Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.
RESUMO
Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.
RESUMO
Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Microscopia Crioeletrônica , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Recombinação Genética , Proteína de Replicação A/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.
Assuntos
Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Fatores de Transcrição/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Cromatina , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/ultraestrutura , Instabilidade Genômica , Humanos , Ligação Proteica/genética , Fatores de Transcrição/genética , Fatores de Transcrição/ultraestruturaRESUMO
BReast Cancer Associated proteins 1 and 2 (BRCA1, -2) and Partner and Localizer of BRCA2 (PALB2) protein are tumour suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. PALB2 coordinates functions of BRCA1 and BRCA2 during homology-directed repair (HDR) and interacts with several chromatin proteins. In addition to protein scaffold function, PALB2 binds DNA. The functional role of this interaction is poorly understood. We identified a major DNA-binding site of PALB2, mutations in which reduce RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the function of RAD51 recombinase. Surprisingly, it possesses the strand exchange activity without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , RNA/metabolismo , Recombinação Genética , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , HumanosRESUMO
Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and recalcitrant. Microbes, however, have evolved the ability to utilize these highly reduced and recalcitrant compounds as a potential source of carbon and energy. Aerobic degradation of aromatics is initiated by oxidizing the aromatic ring, making them more susceptible to cleavage by ring-cleaving dioxygenases. A preponderance of aromatic degradation genes on plasmids, transposons, and integrative genetic elements (and their shuffling through horizontal gene transfer) have lead to the evolution of novel aromatic degradative pathways. This enables the microorganisms to utilize a multitude of aromatics via common routes of degradation leading to metabolic diversity. In this review, we emphasize the exquisiteness and relevance of bacterial degradation of aromatics, interlinked degradative pathways, genetic and metabolic regulation, carbon source preference, and biosurfactant production. We have also explored the avenue of metagenomics, which opens doors to a plethora of uncultured and uncharted microbial genetics and metabolism that can be used effectively for bioremediation.
Assuntos
Bactérias Aeróbias/metabolismo , Genômica , Hidrocarbonetos Aromáticos/metabolismo , Bactérias Aeróbias/genética , Biodegradação Ambiental , Carbono/química , Carbono/metabolismo , Hidrocarbonetos Aromáticos/química , Oxirredução , Tensoativos/metabolismoRESUMO
Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.
Assuntos
Alcaligenes/enzimologia , Hidroxibenzoatos/metabolismo , Pseudomonas/enzimologia , Microbiologia do Solo , Alcaligenes/genética , Alcaligenes/crescimento & desenvolvimento , Alcaligenes/isolamento & purificação , Meios de Cultura , Dioxigenases/metabolismo , Indução Enzimática , Oxigenases de Função Mista/metabolismo , Parabenos/metabolismo , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/isolamento & purificação , Ácido Salicílico/metabolismoRESUMO
Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.
Assuntos
Bacteroides/enzimologia , Esterases/química , Esterases/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Ácidos Cafeicos/metabolismo , Cromatografia em Gel , Ácidos Cumáricos/metabolismo , Cristalografia por Raios X , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Alinhamento de Sequência , Especificidade por Substrato , Xilanos/metabolismoRESUMO
Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dihydroxynaphthalene (1,2-DHN), salicylic acid and catechol. Enzyme activity versus growth profile and heat stability studies suggested the presence of two distinct hydroxylases, namely 1-hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase. 1-Hydroxy-2-naphthoic acid hydroxylase was partially purified (yield 48%, fold 81) and found to be a homodimer with a subunit molecular weight of â¼34 kDa. The enzyme was yellow in color, showed UV-visible absorption maxima at 274, 375 and 445 nm, and fluorescence emission maxima at 527 nm suggested it to be a flavoprotein. The apoenzyme prepared by the acid-ammonium sulfate (2 M) dialysis method was colorless, inactive and lost the characteristic flavin absorption spectra but regained â¼90% activity when reconstituted with FAD. Extraction of the prosthetic group and its analysis by HPLC suggests that the holoenzyme contained FAD. The enzyme was specific for 1-H2NA and failed to show activity with any other hydroxynaphthoic acid analogs or salicylic acid. The K(m) for 1-H2NA in the presence of either NADPH or NADH remained unaltered (72 and 75 µM, respectively), suggesting dual specificity for the coenzyme. The K(m) for FAD was determined to be 4.7 µM. The enzyme catalyzed the conversion of 1-H2NA to 1,2-DHN only under aerobic conditions. These results suggested that 1-hydroxy-2-naphthoic acid hydroxylase is a flavoprotein monooxygenase specific for 1-H2NA and different from salicylate-1-hydroxylase.
Assuntos
Alcaligenes/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Naftóis/metabolismo , Fenantrenos/metabolismo , Alcaligenes/genética , Alcaligenes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Peso Molecular , Especificidade por SubstratoRESUMO
Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the 'phthalic acid' route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of approximately 39 kDa. It required Fe(II) as the cofactor and was specific for 1-hydroxy-2-naphthoic acid (1-H2NA), with K(m) 13.5 microM and V(max) 114 micromol min(-1) mg(-1). 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a K(i) of 116 microM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K(i) of 24 microM. Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O(2) into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradiol-type ring-cleaving dioxygenase.