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Nikorn Thongtip - One of the best experts on this subject based on the ideXlab platform.
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post thawing effects of three cryopreservation diluents on Rusa deer Rusa timorensis spermatozoa
Reproduction in Domestic Animals, 2019Co-Authors: Davide Monaco, Nikorn Thongtip, Eduardo Zappia, Sudsukh Apichaya, Giovanni Michele LacalandraAbstract:The aim of this study was to evaluate home-made and commercial extenders for the cryopreservation of Rusa deer semen. After collection by electroejaculation, six ejaculates were diluted and frozen in TES-based, Tris-based and Triladyl® extenders. Subjective motility, viability, morphology, acrosome integrity and membrane functionality were assessed post-thawing and after 1-hr incubation at 37°C (Thermal stress test). Total and progressive motility, and kinematic parameters were also assessed through CASA system. Post-thawing sperm progressive motility (PM), velocity according to the straight path (VSL) and linearity (LIN) showed significant differences, and higher values were detected for spermatozoa diluted with Triladyl® and TES (p < 0.05) as compared with Tris (PM of Triladyl® 14.7% vs. 3.2% TES and 2.5% Tris; VSL 56 for Triladyl® , 59.2 for TES and 41.7 for Tris; LIN 45.6 for Triladyl® , 52 for TES and 36.5 for Tris). Triladyl® and TES extender led to better post-thawing sperm parameters, but these preliminary results need to be verified through artificial insemination trials.
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Post-thawing effects of three cryopreservation diluents on Rusa deer (Rusa timorensis) spermatozoa.
Reproduction in domestic animals = Zuchthygiene, 2018Co-Authors: Davide Monaco, Eduardo Zappia, Sudsukh Apichaya, Giovanni Michele Lacalandra, Nikorn ThongtipAbstract:The aim of this study was to evaluate home-made and commercial extenders for the cryopreservation of Rusa deer semen. After collection by electroejaculation, six ejaculates were diluted and frozen in TES-based, Tris-based and Triladyl® extenders. Subjective motility, viability, morphology, acrosome integrity and membrane functionality were assessed post-thawing and after 1-hr incubation at 37°C (Thermal stress test). Total and progressive motility, and kinematic parameters were also assessed through CASA system. Post-thawing sperm progressive motility (PM), velocity according to the straight path (VSL) and linearity (LIN) showed significant differences, and higher values were detected for spermatozoa diluted with Triladyl® and TES (p
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study of testicular immunolocalization of inhibin subunits and epididymal histological structures among different antler status in Rusa deer Rusa timorensis
Thai Journal of Veterinary Medicine, 2017Co-Authors: Apichaya Sudsukh, Kazuyoshi Taya, Gen Watanabe, Kentaro Nagaoka, Worawidh Wajjwalku, Nikorn ThongtipAbstract:The number of Rusa deer (Rusa timorensis) raising farms has highly increased in Thailand. Basic data on the reproduction of Rusa deer are essential for improvement in deer farming reproduction and the assisted reproductive technologies (ARTs) of endangered deer species. This study aimed to determine the relation of antler changes to testicular and epididymis histological structure and cellular source of testicular inhibin subunits in Rusa deer. Eighteen Rusa deer testes and epididymis were collected from a slaughterhouse located in Kamphaeng Saen, Nakhon Pathom, Thailand. The male Rusa deer used in this study were separated into three groups depending on antler stages (velvet, intermediate and hard antlers). Histological observations of the testes and epididymis were performed. The testes sections of three different antler stages were immunostained by polyclonal antisera raised against inhibin α, βA and βB subunits. Means (±SEM) of testicular weight and seminiferous tubule diameter in the hard antler group (76.25±9.80 g and 256.10±7.28 μm) were significantly higher than those of the velvet antler group (40.86±7.41 g and 197.25±7.23 μm) (p<0.05). The epididymis of the intermediate and hard antler groups was filled widely with numerous spermatozoa, which, in contrast, were sporadically observed in the velvet antler group. In addition, the inhibin α and inhibin βA subunits were expressed in both Sertoli cells and Leydig cells while the inhibin βB subunit was only expressed in Leydig cells. This study suggests that the Rusa deer testes secrete inhibin in all antler stages. The present study also clearly demonstrates that Rusa deer antler changes are related to testicular weight, seminiferous tubule diameter and epididymal spermatozoa, indicating that antler change is a useful indicator for testicular function in Rusa deer.
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Study of testicular immunolocalization of inhibin subunits and epididymal histological structures among different antler status in Rusa deer (Rusa timorensis)
Thai Journal of Veterinary Medicine, 2017Co-Authors: Apichaya Sudsukh, Kazuyoshi Taya, Kentaro Nagaoka, Worawidh Wajjwalku, Watanabe, Nikorn ThongtipAbstract:The number of Rusa deer (Rusa timorensis) raising farms has highly increased in Thailand. Basic data on the reproduction of Rusa deer are essential for improvement in deer farming reproduction and the assisted reproductive technologies (ARTs) of endangered deer species. This study aimed to determine the relation of antler changes to testicular and epididymis histological structure and cellular source of testicular inhibin subunits in Rusa deer. Eighteen Rusa deer testes and epididymis were collected from a slaughterhouse located in Kamphaeng Saen, Nakhon Pathom, Thailand. The male Rusa deer used in this study were separated into three groups depending on antler stages (velvet, intermediate and hard antlers). Histological observations of the testes and epididymis were performed. The testes sections of three different antler stages were immunostained by polyclonal antisera raised against inhibin α, βA and βB subunits. Means (±SEM) of testicular weight and seminiferous tubule diameter in the hard antler group (76.25±9.80 g and 256.10±7.28 μm) were significantly higher than those of the velvet antler group (40.86±7.41 g and 197.25±7.23 μm) (p
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Annual ovarian activity monitored by the noninvasive measurement of fecal concentrations of progesterone and 17β-estradiol metabolites in Rusa deer (Rusa timorensis)
The Journal of veterinary medical science, 2016Co-Authors: Apichaya Sudsukh, Kazuyoshi Taya, Worawidh Wajjwalku, Watanabe, Ampika Thongphakdee, Nikorn ThongtipAbstract:To clarify the reproductive cycle of female Rusa deer (Rusa timorensis), the fecal concentrations of progesterone and 17β-estradiol metabolites were measured. Fecal samples were collected on a weekly basis for one year (between October, 2012 and September, 2013) from five healthy adult hinds in Thailand. At the beginning of the study, three hinds were pregnant. Two hinds delivered one healthy offspring, and one hind delivered a stillborn calf. The mating period of Rusa hinds in Thailand is from November to April. In pregnant hinds, fecal progesterone metabolite concentration was high in late pregnancy and abruptly declined to the baseline around parturition, suggesting that the placenta secretes a large amount of progesterone. Fecal 17β-estradiol metabolite concentration remained elevated around the day of parturition. Both concentrations of fecal progesterone and 17β-estradiol metabolites in non-lactating hinds were significantly higher than those in lactating hinds, indicating that ovarian activity of lactating hinds is suppressed by the suckling stimulus of fawn during lactation. The present study demonstrated that monitoring of fecal steroid hormones is useful method for assessing ovarian function in this species.
Robert G. Lloyd - One of the best experts on this subject based on the ideXlab platform.
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Rusa holliday junction resolvase dna complex structure insights into selectivity and specificity
Nucleic Acids Research, 2006Co-Authors: Rachel Macmaster, Edward L. Bolt, Robert G. Lloyd, Svetlana E. Sedelnikova, Patrick J. Baker, John B. RaffertyAbstract:We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli Rusa resolvase bound to a duplex DNA substrate that reveals critical protein–DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The Rusa enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, Rusa shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, Rusa does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by Rusa.
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Rusa Holliday junction resolvase: DNA complex structure—insights into selectivity and specificity
Nucleic acids research, 2006Co-Authors: Rachel Macmaster, Edward L. Bolt, Robert G. Lloyd, Svetlana E. Sedelnikova, Patrick J. Baker, John B. RaffertyAbstract:We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli Rusa resolvase bound to a duplex DNA substrate that reveals critical protein–DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The Rusa enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, Rusa shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, Rusa does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by Rusa.
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Rusa proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions.
Molecular microbiology, 2002Co-Authors: Gary J. Sharples, Edward L. Bolt, Robert G. LloydAbstract:The Rusa protein of Escherichia coli is a DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5' to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that Rusa-like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, Rusa is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of Rusa with prophage-like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that Rusa may be of bacterial origin. The Rusa proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence-dependent manner, but the A. aeolicus Rusa shows a different sequence preference (3' to TG) from the E. coli protein (5' to CC), and the r1t Rusa has relaxed sequence dependence, requiring only a single cytosine.
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Substrate Specificity of Rusa Resolvase Reveals the DNA Structures Targeted by RuvAB and RecG In Vivo
Molecular cell, 2002Co-Authors: Edward L. Bolt, Robert G. LloydAbstract:Rusa endonuclease cleaves Holliday junctions by introducing paired strand incisions 5' to CC dinucleotides. Coordinated catalysis is achieved when both subunits of the homodimer interact simultaneously with cleavage sites located symmetrically. This requirement confers Holliday junction specificity. Uncoupled catalysis occurs when binding interactions are disturbed. Genetic studies indicate that uncoupling occurs rarely in vivo, and DNA cleavage is therefore restricted to Holliday junctions. We exploited the specificity of Rusa to identify the DNA substrates targeted by the RuvAB and RecG branch-migration proteins in vivo. We present evidence that replication restart in UV-irradiated cells relies on the processing of stalled replication forks by RecG helicase and of Holliday junctions by the RuvABC resolvasome, and that RuvAB alone may not promote repair.
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Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the Rusa holliday junction resolvase.
Journal of molecular biology, 2000Co-Authors: Edward L. Bolt, Gary J. Sharples, Robert G. LloydAbstract:Holliday junctions are key intermediates in both homologous recombination and DNA repair, and are also formed from replication forks stalled at lesions in the template strands. Their resolution is critical for chromosome segregation and cell viability, and is mediated by a class of small, homodimeric endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active centres are characterised by conserved aspartate/glutamate residues that provide ligands for metal binding. Sequence alignments reveal that they also contain a number of conserved basic residues. We used site-directed mutagenesis to investigate such residues in the Rusa resolvase. Rusa is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to promote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contrast to RuvC, the Rusa resolvase does not interact directly with a branch migration motor, which simplifies analysis of its resolution activity. Catalysis depends on three highly conserved acidic residues (Asp70, Asp72 and Asp91) that define the catalytic centre. We show that Lys76, which is invariant in Rusa sequences, is essential for catalysis, but not for DNA binding, and that an invariant asparagine residue (Asn73) is required for optimal activity. Analysis of DNA binding revealed that Rusa may interact with one face of an open junction before manipulating its conformation in the presence of Mg(2+) as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the first insights into the roles played by basic residues in DNA binding and catalysis by a Holliday junction resolvase.
M. De Garine-wichatitsky - One of the best experts on this subject based on the ideXlab platform.
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Home range and habitat use of introduced Rusa deer (Cervus timorensis russa) in a mosaic of savannah and native sclerophyll forest of new Caledonia
New Zealand Journal of Zoology, 2006Co-Authors: Jérôme Spaggiari, M. De Garine-wichatitskyAbstract:Abstract New Caledonia's unique flora evolved in the absence of ruminant herbivores until the 19th century. Native sclerophyll forests have been drastically reduced in extent, and browsing by introduced Rusa deer is considered a major threat. We carried out a radio‐tracking study on wild Rusa deer on a mosaic of savannahs and sclerophyll forest. The estimated mean home range size was 501 ±33 ha, with little seasonal variation. Deer showed a remarkable site fidelity (the average distance between the site of capture and the geographic centre of activity was 1042 ± 149 m). They used preferentially the sclerophyll forest and flood plains, especially during the dry season. These results confirm that Rusa deer are likely to have a significant impact on the threatened sclerophyll forests, and they give some positive prospects for population control implemented locally.
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The diets of introduced Rusa deer (Cervus timorensis russa) in a native sclerophyll forest and a native rainforest of New Caledonia
New Zealand Journal of Zoology, 2005Co-Authors: M. De Garine-wichatitsky, Yohann Soubeyran, Daniel Maillard, Patrick DuncanAbstract:Abstract New Caledonia has an exceptionally diverse and unique flora, and there is growing concern about the impacts of introduced wild Rusa deer on native forests. The diets of free‐ranging Rusa deer from two native forest sites were studied using rumen content analysis. Samples (n = 61) from a sclerophyll forest site consisted principally of graminoids (64.6 ± 4.4% dry weight), mainly native grass, but the proportion of woody species increased during the dry season. In the rumen samples from the rainforest site (n = 56), woody species were the predominant plants (61.9 ± 3.7% dry weight), and the composition of the diet was more constant across seasons. Most of the food items found could not be identified to species level, but it is estimated that native plants represent 40–60% of Rusa deer diet. Although more work is needed to assess diet preferences and impacts of Rusa deer, we suggest that they represent a potentially important threat to some native plant species in New Caledonia.
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A review of the diet of Rusa Deer Cervus timorensis russa in New Caledonia: Are the endemic plants defenceless against this introduced, eruptive ruminant?
Pacific Conservation Biology, 2003Co-Authors: M. De Garine-wichatitsky, Patrick Duncan, Philippe Chardonnet, Alice Labbe, Bernard Suprin, Daniel MaillardAbstract:Rusa Deer Cervus timorensis russa was introduced to New Caledonia in 1870 from Java, and has colonized the main island of Grande Terre, where it is found in virtually all the terrestrial biotopes. Despite its abundance and its socio-economic importance for New Caledonians, little is known about the diets of the wild deer populations living in contact with native vegetation which has a high degree of endemism and which, until recently, evolved without ruminant herbivores. We collected information on the diet of Rusa Deer in New Caledonia from published and unpublished reports, a questionnaire addressed to experts and preliminary data from browse-surveys. All sources of information suggested that wild Rusa Deer in New Caledonia is a mixed-feeder, and the list of plants consumed included 25 grasses, 15 forbs, 26 trees/shrubs and 12 vines and ferns. Nearly half (49%) of the plants identified as principal and preferred foods were introduced species. Physical defences (spines and thorns) did not seem to deter deer. It is remarkable that many of the avoided plants were native species (56% of the avoided species). Further research is required to confirm these results, but they are relevant to the evolution of plant/herbivore interactions and to the management of deer populations for conservation perspectives in island ecosystems.
Daniel Maillard - One of the best experts on this subject based on the ideXlab platform.
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The diets of introduced Rusa deer (Cervus timorensis russa) in a native sclerophyll forest and a native rainforest of New Caledonia
New Zealand Journal of Zoology, 2005Co-Authors: M. De Garine-wichatitsky, Yohann Soubeyran, Daniel Maillard, Patrick DuncanAbstract:Abstract New Caledonia has an exceptionally diverse and unique flora, and there is growing concern about the impacts of introduced wild Rusa deer on native forests. The diets of free‐ranging Rusa deer from two native forest sites were studied using rumen content analysis. Samples (n = 61) from a sclerophyll forest site consisted principally of graminoids (64.6 ± 4.4% dry weight), mainly native grass, but the proportion of woody species increased during the dry season. In the rumen samples from the rainforest site (n = 56), woody species were the predominant plants (61.9 ± 3.7% dry weight), and the composition of the diet was more constant across seasons. Most of the food items found could not be identified to species level, but it is estimated that native plants represent 40–60% of Rusa deer diet. Although more work is needed to assess diet preferences and impacts of Rusa deer, we suggest that they represent a potentially important threat to some native plant species in New Caledonia.
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A review of the diet of Rusa Deer Cervus timorensis russa in New Caledonia: Are the endemic plants defenceless against this introduced, eruptive ruminant?
Pacific Conservation Biology, 2003Co-Authors: M. De Garine-wichatitsky, Patrick Duncan, Philippe Chardonnet, Alice Labbe, Bernard Suprin, Daniel MaillardAbstract:Rusa Deer Cervus timorensis russa was introduced to New Caledonia in 1870 from Java, and has colonized the main island of Grande Terre, where it is found in virtually all the terrestrial biotopes. Despite its abundance and its socio-economic importance for New Caledonians, little is known about the diets of the wild deer populations living in contact with native vegetation which has a high degree of endemism and which, until recently, evolved without ruminant herbivores. We collected information on the diet of Rusa Deer in New Caledonia from published and unpublished reports, a questionnaire addressed to experts and preliminary data from browse-surveys. All sources of information suggested that wild Rusa Deer in New Caledonia is a mixed-feeder, and the list of plants consumed included 25 grasses, 15 forbs, 26 trees/shrubs and 12 vines and ferns. Nearly half (49%) of the plants identified as principal and preferred foods were introduced species. Physical defences (spines and thorns) did not seem to deter deer. It is remarkable that many of the avoided plants were native species (56% of the avoided species). Further research is required to confirm these results, but they are relevant to the evolution of plant/herbivore interactions and to the management of deer populations for conservation perspectives in island ecosystems.
Edward L. Bolt - One of the best experts on this subject based on the ideXlab platform.
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Rusa holliday junction resolvase dna complex structure insights into selectivity and specificity
Nucleic Acids Research, 2006Co-Authors: Rachel Macmaster, Edward L. Bolt, Robert G. Lloyd, Svetlana E. Sedelnikova, Patrick J. Baker, John B. RaffertyAbstract:We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli Rusa resolvase bound to a duplex DNA substrate that reveals critical protein–DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The Rusa enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, Rusa shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, Rusa does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by Rusa.
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Rusa Holliday junction resolvase: DNA complex structure—insights into selectivity and specificity
Nucleic acids research, 2006Co-Authors: Rachel Macmaster, Edward L. Bolt, Robert G. Lloyd, Svetlana E. Sedelnikova, Patrick J. Baker, John B. RaffertyAbstract:We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli Rusa resolvase bound to a duplex DNA substrate that reveals critical protein–DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The Rusa enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, Rusa shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, Rusa does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by Rusa.
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Rusa proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions.
Molecular microbiology, 2002Co-Authors: Gary J. Sharples, Edward L. Bolt, Robert G. LloydAbstract:The Rusa protein of Escherichia coli is a DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5' to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that Rusa-like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, Rusa is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of Rusa with prophage-like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that Rusa may be of bacterial origin. The Rusa proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence-dependent manner, but the A. aeolicus Rusa shows a different sequence preference (3' to TG) from the E. coli protein (5' to CC), and the r1t Rusa has relaxed sequence dependence, requiring only a single cytosine.
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Substrate Specificity of Rusa Resolvase Reveals the DNA Structures Targeted by RuvAB and RecG In Vivo
Molecular cell, 2002Co-Authors: Edward L. Bolt, Robert G. LloydAbstract:Rusa endonuclease cleaves Holliday junctions by introducing paired strand incisions 5' to CC dinucleotides. Coordinated catalysis is achieved when both subunits of the homodimer interact simultaneously with cleavage sites located symmetrically. This requirement confers Holliday junction specificity. Uncoupled catalysis occurs when binding interactions are disturbed. Genetic studies indicate that uncoupling occurs rarely in vivo, and DNA cleavage is therefore restricted to Holliday junctions. We exploited the specificity of Rusa to identify the DNA substrates targeted by the RuvAB and RecG branch-migration proteins in vivo. We present evidence that replication restart in UV-irradiated cells relies on the processing of stalled replication forks by RecG helicase and of Holliday junctions by the RuvABC resolvasome, and that RuvAB alone may not promote repair.
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Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the Rusa holliday junction resolvase.
Journal of molecular biology, 2000Co-Authors: Edward L. Bolt, Gary J. Sharples, Robert G. LloydAbstract:Holliday junctions are key intermediates in both homologous recombination and DNA repair, and are also formed from replication forks stalled at lesions in the template strands. Their resolution is critical for chromosome segregation and cell viability, and is mediated by a class of small, homodimeric endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active centres are characterised by conserved aspartate/glutamate residues that provide ligands for metal binding. Sequence alignments reveal that they also contain a number of conserved basic residues. We used site-directed mutagenesis to investigate such residues in the Rusa resolvase. Rusa is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to promote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contrast to RuvC, the Rusa resolvase does not interact directly with a branch migration motor, which simplifies analysis of its resolution activity. Catalysis depends on three highly conserved acidic residues (Asp70, Asp72 and Asp91) that define the catalytic centre. We show that Lys76, which is invariant in Rusa sequences, is essential for catalysis, but not for DNA binding, and that an invariant asparagine residue (Asn73) is required for optimal activity. Analysis of DNA binding revealed that Rusa may interact with one face of an open junction before manipulating its conformation in the presence of Mg(2+) as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the first insights into the roles played by basic residues in DNA binding and catalysis by a Holliday junction resolvase.
