The Experts below are selected from a list of 123 Experts worldwide ranked by ideXlab platform
Josette Banroques - One of the best experts on this subject based on the ideXlab platform.
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analyses of the functional regions of dead box rna helicases with deletion and chimera constructs tested in vivo and in vitro
Journal of Molecular Biology, 2011Co-Authors: Olivier Cordin, Monique Doere, Patrick Linder, Josette Banroques, Kyle N TannerAbstract:The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found in nearly all organisms and that are involved in virtually all processes involving RNA. They are characterized by two tandemly linked, RecA-like domains that contain 11 conserved motifs and highly variable amino- and carboxy-terminal flanking sequences. For this reason, they are often considered to be modular multi-domain proteins. We tested this by making extensive BLASTs and sequence alignments to elucidate the minimal functional unit in nature. We then used this information to construct chimeras and deletions of six essential yeast proteins that were assayed in vivo. We purified many of the different constructs and characterized their biochemical properties in vitro. We found that sequence elements can only be switched between closely related proteins and that the carboxy-terminal sequences are important for high ATPase and strand Displacement Activities and for high RNA binding affinity. The amino-terminal elements were often toxic when overexpressed in vivo, and they may play regulatory roles. Both the amino and the carboxyl regions have a high frequency of sequences that are predicted to be intrinsically disordered, indicating that the flanking regions do not form distinct modular domains but probably assume an ordered structure with ligand binding. Finally, the minimal functional unit of the DEAD-box core starts two amino acids before the isolated phenylalanine of the Q motif and extends to about 35 residues beyond motif VI. These experiments provide evidence for how a highly conserved structural domain can be adapted to different cellular needs.
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the newly discovered q motif of dead box rna helicases regulates rna binding and helicase activity
The EMBO Journal, 2004Co-Authors: Olivier Cordin, Kyle N Tanner, Monique Doere, Patrick Linder, Josette BanroquesAbstract:DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs ∼17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group ∼17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis–Menten parameters for the ATPase Activities. We also measured RNA affinities and strand-Displacement Activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
Catherine H. Freudenreich - One of the best experts on this subject based on the ideXlab platform.
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differential requirement of srs2 helicase and rad51 Displacement Activities in replication of hairpin forming cag ctg repeats
Nucleic Acids Research, 2017Co-Authors: Jennifer H.g. Nguyen, David Viterbo, Ranjith P. Anand, Lauren Verra, Laura Sloan, Guy-franck Richard, Catherine H. FreudenreichAbstract:Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.
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Differential requirement of Srs2 helicase and Rad51 Displacement Activities in replication of hairpin-forming CAG/CTG repeats.
Nucleic acids research, 2017Co-Authors: Jennifer H.g. Nguyen, David Viterbo, Ranjith P. Anand, Lauren Verra, Laura Sloan, Guy-franck Richard, Catherine H. FreudenreichAbstract:Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.
Luis G. Brieba - One of the best experts on this subject based on the ideXlab platform.
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Plant Organellar DNA Polymerases Evolved Multifunctionality through the Acquisition of Novel Amino Acid Insertions.
Genes, 2020Co-Authors: Antolín Peralta-castro, Paola L. García-medel, Noe Baruch-torres, Carlos H. Trasviña-arenas, Víctor Juarez-quintero, Carlos M Morales-vazquez, Luis G. BriebaAbstract:The majority of DNA polymerases (DNAPs) are specialized enzymes with specific roles in DNA replication, translesion DNA synthesis (TLS), or DNA repair. The enzymatic characteristics to perform accurate DNA replication are in apparent contradiction with TLS or DNA repair abilities. For instance, replicative DNAPs incorporate nucleotides with high fidelity and processivity, whereas TLS DNAPs are low-fidelity polymerases with distributive nucleotide incorporation. Plant organelles (mitochondria and chloroplast) are replicated by family-A DNA polymerases that are both replicative and TLS DNAPs. Furthermore, plant organellar DNA polymerases from the plant model Arabidopsis thaliana (AtPOLIs) execute repair of double-stranded breaks by microhomology-mediated end-joining and perform Base Excision Repair (BER) using lyase and strand-Displacement Activities. AtPOLIs harbor three unique insertions in their polymerization domain that are associated with TLS, microhomology-mediated end-joining (MMEJ), strand-Displacement, and lyase Activities. We postulate that AtPOLIs are able to execute those different functions through the acquisition of these novel amino acid insertions, making them multifunctional enzymes able to participate in DNA replication and DNA repair.
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Identification of a unique insertion in plant organellar DNA polymerases responsible for 5'-dRP lyase and strand-Displacement Activities: Implications for Base Excision Repair.
DNA repair, 2018Co-Authors: Carlos H. Trasviña-arenas, Paola L. García-medel, Noe Baruch-torres, Antolín Peralta-castro, Francisco J. Cordoba-andrade, Víctor M. Ayala-garcía, Corina Diaz-quezada, José Juan Ordaz-ortiz, Luis G. BriebaAbstract:Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5'-deoxyribose phosphate (5'-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5'-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5'-dRP moiety using their intrinsic lyase and/or strand-Displacement Activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5'-dRP lyase Activities. AtPolIB performs efficient strand-Displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-Displacement activity. Both lyase and strand-Displacement Activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.
Jennifer H.g. Nguyen - One of the best experts on this subject based on the ideXlab platform.
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differential requirement of srs2 helicase and rad51 Displacement Activities in replication of hairpin forming cag ctg repeats
Nucleic Acids Research, 2017Co-Authors: Jennifer H.g. Nguyen, David Viterbo, Ranjith P. Anand, Lauren Verra, Laura Sloan, Guy-franck Richard, Catherine H. FreudenreichAbstract:Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.
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Differential requirement of Srs2 helicase and Rad51 Displacement Activities in replication of hairpin-forming CAG/CTG repeats.
Nucleic acids research, 2017Co-Authors: Jennifer H.g. Nguyen, David Viterbo, Ranjith P. Anand, Lauren Verra, Laura Sloan, Guy-franck Richard, Catherine H. FreudenreichAbstract:Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.
Kyle N Tanner - One of the best experts on this subject based on the ideXlab platform.
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analyses of the functional regions of dead box rna helicases with deletion and chimera constructs tested in vivo and in vitro
Journal of Molecular Biology, 2011Co-Authors: Olivier Cordin, Monique Doere, Patrick Linder, Josette Banroques, Kyle N TannerAbstract:The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found in nearly all organisms and that are involved in virtually all processes involving RNA. They are characterized by two tandemly linked, RecA-like domains that contain 11 conserved motifs and highly variable amino- and carboxy-terminal flanking sequences. For this reason, they are often considered to be modular multi-domain proteins. We tested this by making extensive BLASTs and sequence alignments to elucidate the minimal functional unit in nature. We then used this information to construct chimeras and deletions of six essential yeast proteins that were assayed in vivo. We purified many of the different constructs and characterized their biochemical properties in vitro. We found that sequence elements can only be switched between closely related proteins and that the carboxy-terminal sequences are important for high ATPase and strand Displacement Activities and for high RNA binding affinity. The amino-terminal elements were often toxic when overexpressed in vivo, and they may play regulatory roles. Both the amino and the carboxyl regions have a high frequency of sequences that are predicted to be intrinsically disordered, indicating that the flanking regions do not form distinct modular domains but probably assume an ordered structure with ligand binding. Finally, the minimal functional unit of the DEAD-box core starts two amino acids before the isolated phenylalanine of the Q motif and extends to about 35 residues beyond motif VI. These experiments provide evidence for how a highly conserved structural domain can be adapted to different cellular needs.
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the newly discovered q motif of dead box rna helicases regulates rna binding and helicase activity
The EMBO Journal, 2004Co-Authors: Olivier Cordin, Kyle N Tanner, Monique Doere, Patrick Linder, Josette BanroquesAbstract:DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs ∼17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group ∼17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis–Menten parameters for the ATPase Activities. We also measured RNA affinities and strand-Displacement Activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.