The Experts below are selected from a list of 162 Experts worldwide ranked by ideXlab platform
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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An RNA Ligase from Deinococcus radiodurans
The Journal of biological chemistry, 2004Co-Authors: Alexandra Martins, Stewart ShumanAbstract:Although DNA repair pathways have been the focus of much attention, there is an emerging appreciation that distinct pathways exist to maintain or manipulate RNA structure in response to breakage events. Here we identify an RNA Ligase (DraRnl) from the radiation-resistant bacterium Deinococcus radiodurans. DraRnl seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA: DNA hybrid, but it cannot seal 3'-OH/5'-PO4 DNA nicks. The specificity of DraRnl arises from a requirement for RNA on the 3'-OH side of the nick. DraRnl is a 342-amino acid monomeric protein with a distinctive structure composed of a C-terminal adenylyltransferase domain linked to an N-terminal module that resembles the OB-fold of phenylalanyl-tRNA synthetases. RNA sealing activity was abolished by mutation of the predicted lysine adenylylation site (Lys-165) in the C-terminal domain and was reduced by an order of magnitude by deletion of the N-terminal OB module. Our findings highlight the existence of an RNA repair capacity in bacteria and support the hypothesis that contemporary DNA Ligases, RNA Ligases, and RNA capping enzymes evolved by the fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.
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Structure and Mechanism of RNA Ligase
Structure, 2004Co-Authors: Li Kai Wang, Christopher D. Lima, Stewart ShumanAbstract:T4 RNA Ligase 2 (Rnl2) exemplifies an RNA Ligase family that includes the RNA editing Ligases (RELs) of Trypanosoma and Leishmania . The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3′-OH and 5′-PO 4 RNA ends by Rnl2, but not for Ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA Ligase domain. We report the 1.9 A crystal structure of the Ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA Ligases, DNA Ligases, and mRNA capping enzymes.
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Structure-function analysis of T4 RNA Ligase 2.
Journal of Biological Chemistry, 2003Co-Authors: Shenmin Yin, Stewart ShumanAbstract:Abstract Bacteriophage T4 RNA Ligase 2 (Rnl2) exemplifies a polynucleotide Ligase family that includes the trypanosome RNA-editing Ligases and putative RNA Ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like Ligases, DNA Ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu34 and Asn40, that flank the active site lysine nucleophile (Lys35) and are unique to the Rnl2-like Ligase family.
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Bacteriophage T4 RNA Ligase 2 (gp24.1) exemplifies a family of RNA Ligases found in all phylogenetic domains
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Stewart ShumanAbstract:RNA Ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA Ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA Ligase and trypanosome RNA-editing Ligases. Here, we identified another RNA Ligase from the bacterial domain—a second RNA Ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through Ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the Ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA Ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct Ligase family, defined by variant motifs, that includes the trypanosome-editing Ligases and a group of putative RNA Ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.
José-antonio Daròs - One of the best experts on this subject based on the ideXlab platform.
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viroid RNA redirects host dna Ligase 1 to act as an RNA Ligase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: María-Ángeles Nohales, Ricardo Flores, José-antonio DaròsAbstract:Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246–401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNAse presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA Ligase 1, converting it to an RNA Ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA Ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95–G96 and containing 5′-phosphomonoester and 3′-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA Ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA Ligase to act as RNA-dependent RNA polymerase and RNA Ligase, respectively.
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A chloroplastic RNA Ligase activity analogous to the bacterial and archaeal 2´-5' RNA Ligase.
RNA Biology, 2012Co-Authors: Diego Molina-serrano, Ricardo Flores, José-antonio Daròs, Jorge Marqués, María-Ángeles Nohales, María-Ángeles Nohales, Ricardo Flores, José-antonio DaròsAbstract:Bacteria and archaea contain a 2’-5′ RNA Ligase that seals in vitro 2’,3′-cyclic phosphodiester and 5′-hydroxyl RNA termini, generating a 2’,5′-phosphodiester bond. In our search for an RNA Ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA Ligase activity whose properties resemble those of the bacterial and archaeal 2’-5′ RNA Ligase. The spinach chloroplastic RNA Ligase recognizes the 5′-hydroxyl and 2’,3′-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2’,5′-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear i...
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A chloroplastic RNA Ligase activity analogous to the bacterial and archaeal 2´-5' RNA Ligase.
RNA biology, 2012Co-Authors: Diego Molina-serrano, Ricardo Flores, José-antonio Daròs, Jorge Marqués, María-Ángeles Nohales, María-Ángeles Nohales, Ricardo Flores, José-antonio DaròsAbstract:Bacteria and archaea contain a 2'-5' RNA Ligase that seals in vitro 2',3'-cyclic phosphodiester and 5'-hydroxyl RNA termini, generating a 2',5'-phosphodiester bond. In our search for an RNA Ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA Ligase activity whose properties resemble those of the bacterial and archaeal 2'-5' RNA Ligase. The spinach chloroplastic RNA Ligase recognizes the 5'-hydroxyl and 2',3'-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2',5'-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear initial substrate. Even if its involvement in viroid replication seems unlikely, the identification of a 2'-5' RNA Ligase activity in higher plant chloroplasts, with properties very similar to an analogous enzyme widely distributed in bacterial and archaeal proteomes, is intriguing and suggests an important biological role so far unknown.
Anna Gudny Hermannsdottir - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of a thermostable RNA Ligase 1 from a thermus scotoductus bacteriophage ts2126 with good single stranded dna ligation properties
Nucleic Acids Research, 2005Co-Authors: Thorarinn Blondal, Sigridur Hjorleifsdottir, Olafur F. Fridjonsson, Arnthor Aevarsson, Sigurlaug Skirnisdottir, Audur Thorisdottir, Unnur Unnsteinsdottir, Sveinn Ernstsson, Jon Oskar Wheat, Anna Gudny HermannsdottirAbstract:We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA Ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA Ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA Ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA Ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.
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Discovery and characterization of a thermostable bacteriophage RNA Ligase homologous to T4 RNA Ligase 1
Nucleic acids research, 2003Co-Authors: Thorarinn Blondal, Sigridur Hjorleifsdottir, Olafur F. Fridjonsson, Arnthor Aevarsson, Sigurlaug Skirnisdottir, Anna Gudny Hermannsdottir, Gudmundur O. Hreggvidsson, Albert Vernon Smith, Jakob K. KristjanssonAbstract:Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA Ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA Ligase 1 has a temperature optimum of 60–64°C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA Ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.
Thorarinn Blondal - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of a thermostable RNA Ligase 1 from a thermus scotoductus bacteriophage ts2126 with good single stranded dna ligation properties
Nucleic Acids Research, 2005Co-Authors: Thorarinn Blondal, Sigridur Hjorleifsdottir, Olafur F. Fridjonsson, Arnthor Aevarsson, Sigurlaug Skirnisdottir, Audur Thorisdottir, Unnur Unnsteinsdottir, Sveinn Ernstsson, Jon Oskar Wheat, Anna Gudny HermannsdottirAbstract:We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA Ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA Ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA Ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA Ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.
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Discovery and characterization of a thermostable bacteriophage RNA Ligase homologous to T4 RNA Ligase 1
Nucleic acids research, 2003Co-Authors: Thorarinn Blondal, Sigridur Hjorleifsdottir, Olafur F. Fridjonsson, Arnthor Aevarsson, Sigurlaug Skirnisdottir, Anna Gudny Hermannsdottir, Gudmundur O. Hreggvidsson, Albert Vernon Smith, Jakob K. KristjanssonAbstract:Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA Ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA Ligase 1 has a temperature optimum of 60–64°C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA Ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.
María-Ángeles Nohales - One of the best experts on this subject based on the ideXlab platform.
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viroid RNA redirects host dna Ligase 1 to act as an RNA Ligase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: María-Ángeles Nohales, Ricardo Flores, José-antonio DaròsAbstract:Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246–401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNAse presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA Ligase 1, converting it to an RNA Ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA Ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95–G96 and containing 5′-phosphomonoester and 3′-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA Ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA Ligase to act as RNA-dependent RNA polymerase and RNA Ligase, respectively.
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A chloroplastic RNA Ligase activity analogous to the bacterial and archaeal 2´-5' RNA Ligase.
RNA Biology, 2012Co-Authors: Diego Molina-serrano, Ricardo Flores, José-antonio Daròs, Jorge Marqués, María-Ángeles Nohales, María-Ángeles Nohales, Ricardo Flores, José-antonio DaròsAbstract:Bacteria and archaea contain a 2’-5′ RNA Ligase that seals in vitro 2’,3′-cyclic phosphodiester and 5′-hydroxyl RNA termini, generating a 2’,5′-phosphodiester bond. In our search for an RNA Ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA Ligase activity whose properties resemble those of the bacterial and archaeal 2’-5′ RNA Ligase. The spinach chloroplastic RNA Ligase recognizes the 5′-hydroxyl and 2’,3′-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2’,5′-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear i...
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A chloroplastic RNA Ligase activity analogous to the bacterial and archaeal 2´-5' RNA Ligase.
RNA biology, 2012Co-Authors: Diego Molina-serrano, Ricardo Flores, José-antonio Daròs, Jorge Marqués, María-Ángeles Nohales, María-Ángeles Nohales, Ricardo Flores, José-antonio DaròsAbstract:Bacteria and archaea contain a 2'-5' RNA Ligase that seals in vitro 2',3'-cyclic phosphodiester and 5'-hydroxyl RNA termini, generating a 2',5'-phosphodiester bond. In our search for an RNA Ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA Ligase activity whose properties resemble those of the bacterial and archaeal 2'-5' RNA Ligase. The spinach chloroplastic RNA Ligase recognizes the 5'-hydroxyl and 2',3'-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2',5'-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear initial substrate. Even if its involvement in viroid replication seems unlikely, the identification of a 2'-5' RNA Ligase activity in higher plant chloroplasts, with properties very similar to an analogous enzyme widely distributed in bacterial and archaeal proteomes, is intriguing and suggests an important biological role so far unknown.
