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Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C–80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.
Citation: Gardner AF, Guan C, Jack WE (2011) Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2. PLoS ONE 6(8): e23668. https://doi.org/10.1371/journal.pone.0023668
Editor: Jean-Pierre Vartanian, Institut Pasteur, France
Received: June 2, 2011; Accepted: July 22, 2011; Published: August 17, 2011
Copyright: © 2011 Gardner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by New England Biolabs, Inc (www.neb.com). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors are employed by New England Biolabs, Inc. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. This study was conducted in the Research Department and is not currently intended for product development.
Holliday junction resolving enzymes are structure-specific endonucleases that catalyze key steps during DNA homologous recombination and replication [1]. Resolving enzymes have been identified in all domains of life including bacteria (RuvC), archaea (Hjc), and eukarya (Human GEN1) [2]. Resolving enzymes have attracted intense interest as a model to understand the molecular basis for substrate recognition and cleavage of four-way junctions [2]. Variations in resolving enzyme sequence bias, cleavage pattern, and substrate specificity suggest that a variety of mechanisms have evolved to cleave four-way junctions [3], [4], [5], [6], [7], [8], [9], [10], [11]. Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C–80°C) and acidity (pH 3) and encodes a 14 kD Holliday junction resolving enzyme (SIRV2 Hjr). Hjr protein sequences are conserved among rudiviruses including Acidianus Rod-Shaped virus 1 (ARV1), Stygioglobus rod-shaped virus (SRV), and Sulfolobus islandicus rod-shaped viruses 1 (SIRV1) and 2 (SIRV2), and have been proposed as key enzymes in rudivirus genome replication [12]. Specifically, during the last stage of SIRV2 replication, multiple double-stranded SIRV2 genomes are catenated. At the junctions between genome monomers, opposing inverted terminal repeats can be extruded to form hairpin four-way junctions. SIRV2 Hjr is proposed to introduce symmetrical nicks across this junction to resolve the concatamers, producing monomer copies with linear hairpin ends [12]. Consistent with its proposed biological role, SIRV2 Hjr was previously shown to cleave four-way junctions in vitro [13], [14]. SIRV2 Hjr is related to the well-studied resolving enzymes from Sulfolobus, Hje and Hjc [4], [9], [15], [16], [17], [18], [19]. Sulfolobus Hje and Hjc are homodimeric enzymes that recognize and cleave four-way junctions by paired nicks on four-way junction arms [4], [18]. Even though a significant amount of data is available on Sulfolobus resolving enzymes, much less is known about the molecular mechanism of SIRV2 Hjr specificity and cleavage. Therefore, this study demonstrates a unique SIRV2 Hjr four-way junction cleavage pattern, the minimal requirements for four-way junction cleavage, and substrate specificity. Based on the biochemical analysis of SIRV2 Hjr, this study also addresses characteristics that support a role for SIRV2 Hjr in resolution following genome replication.
Materials and Methods
"Hjr" will be used throughout to refer to the product of the allele from S. islandicus SIRV2 Hjr (or the codon-optimized allele described herein), and "MBP-Hjr" will refer to its fusion to Maltose Binding Protein below. Where necessary for clarity, the attribution will be expanded.
All restriction endonucleases, modifying enzymes, DNA polymerases, nucleotides, DNA ladders, and expression vectors were from New England Biolabs. Purified Sulfolobus solfataricus Holliday junction endonuclease (Sso Hje) was kindly provided by Dr. Malcolm White, University of St. Andrew's, UK.
E. coli strains for cloning (NEB 5 alpha) and expression (NEB Turbo and NEB T7 Express) were from New England Biolabs.
MBP-SIRV2 Hjr gene synthesis, cloning and purification
To improve protein expression, a synthetic Hjr gene was codon optimized to reflect the codon usage of E. coli rather than the native S. islandicus. Hjr gene was synthesized by PCR amplification of overlapping oligonucleotides [20].
To assemble a template for Hjr gene synthesis, an equimolar amount (1 µM) of each overlapping oligonucleotide (Table S1) was combined in 1× Standard Taq Buffer (10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2) and then serially diluted by two-fold. PCR reactions (50 µL) were assembled as follows: 1× Phusion Master Mix (containing dNTPs, HF reaction buffer, and Phusion DNA polymerase), 0.5 µM Forward Primer (Table S1, primer 1), 0.5 µM Reverse Primer (Table S1, primer 10), and Hjr gene synthesis oligonucleotide template mixtures. Reactions were cycled in a PCR instrument (98°C 2 minutes followed by 25 cycles of 98°C 10 seconds, 65°C 15 seconds, 72°C for 30 seconds, followed by a final extension step at 72°C for 30 seconds). A band corresponding to the Hjr gene (405 bp) was gel purified. The Hjr codon optimized PCR product was cloned into expression vector pMAL-c4X (New England Biolabs) digested with XmnI to create a construct (pEPD) encoding an N-terminal Maltose Binding Protein (MBP) – SIRV2 Hjr fusion protein. The sequence of plasmid pEPD was verified by DNA sequencing.
For MBP-Hjr expression and purification, NEB Turbo E. coli was transformed with plasmid pEPD. A 1 liter NEB Turbo E. coli/pEPD culture was grown at 37°C to mid-log phase (OD600 = 0.5), whereupon protein expression was induced by addition of 0.4 mM IPTG. Cells were then incubated at 37°C for five hours, and were collected by centrifugation. The cell pellet was suspended in 0.2 L Buffer A (20 mM TrisHCl, pH 7.5, 0.2 M NaCl, 1 mM EDTA) and lysed by sonication. Cell debris was removed by centrifugation and the supernatant applied to a 15 mL amylose column. The column was washed with 0.15 L Buffer A. MBP-SIRV2 Hjr was eluted with 30 mL Buffer A containing 10 mM maltose. MBP-Hjr purification was monitored by 4–20% SDS-PAGE analysis. Fractions containing MBP-Hjr were pooled, dialysed against storage buffer (0.1 M KCl, 10 mM Tris-HCl, pH 7.4 @ 25°C, 1 mM dithiothreitol, 0.1 mM EDTA, 50% Glycerol) and stored at −20°C. A portion of MBP-Hjr was proteolysed by Factor Xa protein to separate the MBP binding domain from SIRV2 Hjr and not further purified. Activity at 55°C was comparable between the intact and proteolyzed proteins, so the MBP-Hjr fusion was used in most experiments. However, it should be noted that even though no apparent differences in overall activity were observed using the MBP-Hjr fusion, steps in the reaction pathway may be influenced by the presence of the MBP-fusion.
MBP-Sulfolobus islandicus Holliday junction endonuclease (Sis Hje) gene synthesis, cloning and purification
The gene encoding a Holliday junction endonuclease from Sulfolobus islandicus strain Y.N.15.51 (Sis Hje) [21] was synthesized using the methods described above using the overlapping oligonucleotides presented in Table S1. Sis Hje E. coli codon optimized PCR product was cloned into expression vector pMAL-c4X digested with XmnI to create a construct encoding an N-terminal Maltose Binding Protein (MBP) – Sis Hje fusion protein.
SIRV2gp26 gene synthesis, cloning and purification
A gene encoding the SIRV2gp26 coat protein was synthesized using the methods described above, with the overlapping oligonucleotides listed in Table S1. The SIRV2gp26 E. coli codon optimized PCR product was cloned into expression vector pMAL-c4X digested with XmnI to create a construct encoding an N-terminal Maltose Binding Protein (MBP) – SIRV2gp26 fusion protein. A portion of the MBP-SIRV2gp26 was treated with Factor Xa protease to separate MBP and SIRV2gp26 and heated to 65°C for 20 minutes to inactive the protease.
DNA substrates
Two plasmids containing hairpin four-way junctions were constructed to assay resolving enzyme activity. pUC(AT) is a derivative of pUC19 containing an inverted repeat of twenty A and T dinucleotides ((AT)20) between the EcoRI and PstI sites that forms a hairpin four-way junction upon supercoiling [22], [23] (Figure S1A). Plasmid pEMM2 is derived from pNEB206A (NEB, Ipswich, MA) and contains an insert corresponding to the expected four-way junction formed between SIRV2 genome dimers during genome replication (Figure S1B). pEMM2 was constructed by annealing overlapping SIRV2 four-way junction oligonucleotides in 1× Standard Taq Buffer (10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2). This oligonucleotide cassette with 3′ overhangs was ligated to complementary ends on pNEB206A vector linearized by XbaI and Nt. BbvCI (New England Biolabs) to create pEMM2. The correct pEMM2 sequence was confirmed by DNA sequencing.
Synthetic oligonucleotide substrates were also used to characterize resolving enzyme specificity and activity. Four-way Junction 3 (J3) was constructed by annealing strands (25 µM each) b, h, r, and x in 1× Standard Taq Buffer. In addition, fluorescently labeled J3 four-way junctions were prepared by annealing one 6-carboxyfluorescein (FAM)-labeled strand and three unlabeled strands. A SIRV2 four-way junction was constructed by annealing strands (25 µM each) 1, 2, 3, and 4.
In addition to four-way junction DNA, Hjr activity was assayed on alternate DNA structures, including single- and double-stranded DNA, bulged DNA, hairpin, and three-strand Holliday-like junctions. Schematics representing the DNA structures used in these experiments are depicted in Figure 1. Oligonucleotides for DNA substrates are listed in Table S2. Double-stranded DNA was formed by annealing FAM-labeled top strand to an unlabeled complement in 1× Standard Taq Buffer. A FAM-labeled hairpin DNA mimicking the SIRV2 genome end was formed by self-annealing. Heteroduplex bulged DNA (25 µM stock) was constructed by annealing two oligonucleotides in 1× Standard Taq Buffer to create an unpaired central region flanked by complementary base pairing. A three-strand Holliday-like junction was prepared by annealing four-way junction J3 strands (FAM)-b, h, r.
Substrates (A: single-stranded DNA, B: double-stranded DNA, C: heteroduplex double-stranded DNA, D: hairpin DNA, E: four-way junction J3, F: three-way Holliday-like junction) were constructed as described in Materials & Methods and 5′ labeled with fluorescence for detection (as indicated by a black circle). Distilled water (-), Hjr (S) (20 nM) or T7 endonuclease I (T7) (7.5 nM) was incubated with 100 nM substrate in 1× ThermoPol Buffer at 55°C for 30 minutes. Reaction products were separated by 20% denaturing PAGE and fluorescence detected by a GE Typhoon scanner.
Assays for resolving enzyme activity
Four-way junction resolution was monitored by cleavage of either four-way junction containing plasmids (pUC(AT) or pEMM2) or fluorescently labeled synthetic four-way junctions. Typically in a 50 µL reaction, plasmids (11 nM) were incubated with 20 nM resolving enzyme in 1× ThermoPol Buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8 @ 25°C) at 55°C for one hour. Reaction products were separated by agarose gel electrophoresis. Synthetic four-way junctions were constructed as described above and one strand was fluorescently labeled on its 5′ end. In a 20 µL reaction, a synthetic four-way junction (J3 or SIRV2 at 100 nM) was incubated with 20 nM resolving enzyme in 1× ThermoPol Buffer (unless otherwise noted) at 55°C for one hour. FAM-labeled 16-mer and 18-mer were size standards during electrophoresis (Table S2). Reaction products were separated by 20% denaturing PAGE and quantified using the fluorescence detected by a GE Typhoon scanner.
Requirements for SIRV2 Hjr cleavage
Hjr cleavage activity was assayed with synthetic DNA four-way junction J3 under a variety of reaction conditions to determine requirements for cleavage. Four-way junction J3 (FAM-labeled on strand b) (100 nM) and MBP-Hjr (20 nM) were incubated in reaction buffer at 55°C for 30 minutes. Reaction buffers varied in pH (4–10), divalent cation (MgCl2, MnCl2, ZnSO4, CaCl2, CoCl2), or concentrations of NaCl (0–500 mM), NH4SO4 (0–200 mM), or MgCl2 (0–100 mM). Sodium acetate (10 mM) was used in the pH range of 4.0–6.0 and TrisHCl (10 mM) was used in the pH range of 7.0–10. Reactions were halted by addition of 50% formamide and 5 mM EDTA. Reaction products were separated by 20% denaturing PAGE and fluorescence detected on a GE Typhoon scanner.
Hjr substrate specificity
To determine Hjr substrate specificity, a panel of DNA substrates including single- and double-stranded DNA, bulged DNA, hairpin, and three-strand Holliday-like junctions were prepared as described above. MBP-Hjr (20 nM) or T7 endonuclease I (7.5 nM) (New England Biolabs) was incubated with 0.10 µM DNA in 1× ThermoPol Buffer at 55°C for 30 minutes. Reaction products were separated by 20% denaturing PAGE and quantified using the fluorescence detected by a GE Typhoon scanner.
Characterization of Hjr four-way junction cleavage products
In a 0.10 mL reaction, pUC(AT) (25 nM) was incubated with 20 nM MBP-Hjr in 1× ThermoPol Buffer at 55°C. Linearized products were gel purified by QiaPrep PCR purification kit (Qiagen) and eluted in 0.10 mL of EB buffer. Linearized products (50 µL) were treated with 400 U T4 DNA ligase in 1× T4 DNA ligase buffer for one hour at room temperature to seal hairpin nicks. Lambda exonuclease treatment was carried out in a 30 µL reaction by mixing 3 µL 10× lambda exonuclease buffer, 5 U lambda exonuclease and 26 µL of linearized products, and incubated at 37°C for one hour to degrade DNA with free 5′ termini. Reaction products were separated by agarose electrophoresis.
Protein extracts of SIRV2-infected S. islandicus were prepared from 0.25–0.5 L cultures after concentration of the cells by centrifugation, suspension of the cells in 25 mL of 150 mM NaCl, 20 mM TrisHCl, pH 7.5, and 1 mM EDTA and lysis by sonication. Cell debris was removed by centrifugation and the supernatant centrifuged a second time to further remove cell debris. The resulting clarified cell-free lysate was used for further studies. In a 0.5 mL reaction, 10 µg MBP-Hjr and incubated with ∼1 mg SIRV2-infected S. islandicus extract at 4°C for 16 hours with shaking in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 @ 25°C). Anti-MBP magnetic beads were added and affinity complexes were magnetically separated and washed five times with 1.0 mL of NEBuffer 3 to elute non-specifically bound protein. The remaining specific protein complexes were eluted by boiling in 1× SDS-PAGE loading buffer for 5 minutes and analyzed by SDS-PAGE.
Identification of interacting proteins by Mass Spectrometry
Proteins eluted from MBP-Hjr capture experiments were digested into peptides with trypsin and run on an LC/MS-MS for peptide analysis at the New England Biolabs Proteomic Facility. Peptide masses were compared to a database of S. islandicus strain Y.N.15.51 (accession: NC_012623 [21]) and SIRV2 protein sequences using Spectrum Mill software (Agilent Technologies). Peptides that did not exactly match the amino acid sequences in the database, had alternatively charged states, or were modified by phosphorylation or glycosylation were not scored as positives. The sequence of host S. islandicus LAL 14/1 is not publicly available, forcing the use of data from closely related strains, so the list of identified proteins is expected to be an underestimate of true positive interactors in the actual extract. Data was filtered for quality with an MS/MS Score cut off of 15. Proteins from S. islandicus or SIRV2 were identified by sequence comparison using a BLAST [24] similarity score cut off of E<0.1. Interacting proteins identified by mass spectrometry were tabulated and grouped according to their presumed functional role.
Interaction between Hjr and SIRV2gp26 coat protein
To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro, anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs). Bead complexes were mixed thoroughly and incubated at 4°C with shaking for 1 hour. A magnet was applied and supernatant was decanted and bead complexes were washed 5 times with 1× NEBuffer 3. These MBP-Hjr affinity beads (10 µg) were incubated with approximately 10 µg SIRV2gp26 prepared as described earlier at 25°C for 2 hours with shaking in 0.5 mL 1× NEBuffer 3. Affinity complexes were separated by a magnet and washed five times with 1.0 mL of NEBuffer 3 to remove any non-specifically bound protein. Protein complexes were eluted by boiling in 1× SDS-PAGE loading buffer for 5 minutes and analyzed by SDS-PAGE. Background binding was assessed using MBP affinity beads prepared with unfused MBP (anti-MBP::MBP5).
SIRV2 Hjr cleaves four-way junction but not branched DNA
Previously described viral resolving enzymes cleave a variety of branched DNA structures formed during replication [11], [25], [26]. To test if Hjr substrate requirements parallel those of other viral resolving enzymes, Hjr and bacteriophage T7 endonuclease I activities on the panel of DNA substrates depicted in Figure 1 were compared. In contrast to T7 endonuclease I, Hjr only cleaves four-way junction DNA and is inactive on single- or double-stranded DNA, hairpin DNA, heteroduplex loops (bulges), and three-way Holliday-like junctions (Figure 1). S. solfataricus Hje shows a similarly narrow substrate range and only cleaves four-way junction DNA structures [9].
Hjr activity on four-way junction DNA
To identify DNA structural elements and sequences required for Hjr cleavage, activity on a variety of four-way junction DNAs was examined. First, Hjr was tested on plasmid substrates containing junctions that resemble hairpin four-way junction substrates formed during SIRV2 replication in vivo. Plasmid pUC(AT) contains an AT-rich hairpin four-way junction and has been used as a substrate to study T7 endonuclease I and fowlpox resolving enzyme (Figure S1A) [23], [27]. Plasmid pEMM2 includes a hairpin four-way junction that mimics the sequence formed at the SIRV2 concatamer junctions during replication (Figure S1B). Hjr cleaves the pUC(AT) four-way junction to convert closed circular pUC(AT) to a linear form (Figure 2A). Hjr also cleaves the four-way junction structure in plasmid pEMM2 to a linear form (data not shown). The Hjr-cleavage site was mapped by restriction fragment length analysis using XmnI and HindIII and shown to be specific for the DNA four-way junction (Figure S2). Relaxed nicked and linear pUC(AT) and pEMM2 are not substrates, presumably because the four-way junctions do not form in the absence of supercoiling (data not shown). In addition, plasmids lacking four-way junctions are also not substrates (data not shown).
(A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.
Hjr cleavage was then tested on a synthetic four-way junction designed to mimic possible structures formed during SIRV2 replication. The SIRV2 cruciform sequence allows four-way junction motion along the duplex and as the four-way junction migrates along the substrate, the crossover points may vary. The Hjr dimer recognizes four-way junction structures and makes a nick on pairs of four-way junction strands [2]. Hjr nicks strands 1 and 3 (Lanes 1, 3) at three paired sites and makes two paired nicks on the strands 2 and 4 of SIRV2 four-way junction DNA (Lanes 2, 4) (Figure 2B, C). The observed multiple cleavage sites could reflect different four-way junction configurations due to migration, each cleaved at a fixed distance from a crossover point but at different positions relative to the end or alternatively multiple cleavage sites at a single crossover point.
After initial characterization of Hjr activity with the mobile SIRV2 four-way junction, an alternate well-characterized fixed four-way junction DNA substrate (Junction 3 (J3)) was used for further detailed characterization. By utilizing the well-defined junction J3 as a substrate for Hjr cleavage, comparisons can be made to other resolving enzyme cleavage patterns described in the literature that use the same J3 substrate [9], [28], [29]. J3 is composed of four hybridized DNA strands: two DNA strands are continuously stacked and the other two strands base pair with one continuous strand then switch to base pair with the other continuous strand on the adjacent helix (Figure 3A) [30]. The pair of DNA strands that maintain base stacking through the junction are referred to as the continuous strands (strands h and x) while strands that exchange between helices are the exchange strands (strands b and r) (Figure 3A), and bases at the junction are stacked with different strands on the two faces [31]. Local sequence properties change the probability of adopting the stacking switch. The four-way junction J3 favors an isoform conformation with strands h and x as continuous strands and strands b and r as exchange strands [31].
(A) Resolving enzyme cleavage sites are shown on a schematic representation of four-way junction J3 with continuous strands colored blue (x) and black (h) and exchange strands colored red (b) and green (r). Sso Hje and Sis Hje cleave four-way junction J3 junction two base pairs 3′ from the junction in a symmetrical fashion on the h and x continuous strands while Hjr cleaves two base pairs 3′ from the junction in a symmetrical fashion on the b and r exchange strands. (B) Four-way junction J3 DNA (100 nM) 5′ FAM-labeled either on the h, b, r, x strand was incubated with 20 nM S. solfataricus (Sso Hje), S. islandicus Hje (Sis Hje), or Hjr in 1× ThermoPol Buffer at 55°C for 60 minutes. Reaction products were separated by denaturing 20% PAGE. S and P represent substrate (34-mer) and product (18-mer) bands, respectively.
Four-way junction J3 was fluorescently labeled on either the b, h, r, or x strand. Both Sso and Sis Hje cleave preferentially on the continuous strands (h, x) of the J3 four-way junction (Figure 3B). In contrast, Hjr preferentially cleaves the exchange strands (b, r) of this same four-way junction (Figure 3B).
Requirements for SIRV2 Hjr four-way junction cleavage
The reaction conditions for Hjr cleavage were investigated in detail using the synthetic J3 four-way junction. Despite the acidic growth environment, the internal pH of Sulfolobus islandicus is neutral. The pH optimum for Hjr activity reflects its native cytosolic environment with optimal activity between pH 7 to 9, partial activity at pH 6 and 10 (80% or 70% activity, respectively) and no activity below pH 5 (Figure S3A). Consistent with previous studies, a divalent cation (MgCl2 or MnCl2) is required for resolving enzyme activity (Figure S3C) [28]. Hjr activity is optimal between 0.5 and 20 mM MgCl2 (Figure S3B). Hjr is minimally active with CaCl2 and not active with cofactors ZnSO4 or CoCl2 (Figure S3C). NaCl or (NH4)2SO4 are not required for Hjr activity and do not stimulate cleavage (Figure S3D, E). However, Hjr activity is inhibited by higher concentrations of NaCl (>250 mM) and (NH4)2SO4 (>10 mM) (Figure S3D, E).
SIRV2 Hjr four-way hairpin junction cleavage results in linear DNA with hairpin termini
The last stage of SIRV2 replication is envisioned as a concatamer joined by a hairpin four-way junction. Hjr is proposed to introduce symmetrical nicks across this junction to resolve the concatamers, producing single molecules with linear hairpin ends [12]. To test this model, a plasmid-encoded hairpin four-way junction was cut with SIRV2 and the nature of the ends was determined as schematically depicted in Figure 4A. First, the four-way junction containing plasmid, pUC(AT), was cleaved with Hjr to generate a linear fragment and then incubated with buffer alone or with T4 DNA ligase (Figure 4A). Lambda exonuclease was added to digest DNA with free 5′ ends. As predicted, linear fragments are sensitive to lambda exonuclease digestion in the absence of ligation due to free 5′ ends (Figure 4B). However, linear fragments treated with T4 DNA ligase are resistant to lambda exonuclease cleavage suggesting that ends are nicked hairpins that can be ligated to form contiguous covalently closed linear DNA. Therefore, these data support a model in which Hjr cleaves genome concatamers at hairpin four-way junctions to produce single genome copies having nicked hairpin ends that can then be sealed by DNA ligase to form covalently closed molecules.
(A) Hjr was used to cleave the hairpin four-way junction containing plasmid, pUC(AT), to generate linear fragments with nicked hairpin ends. The linear fragments were then incubated with either buffer alone or T4 DNA ligase to seal nicks. Lambda exonuclease (gray shape) was then added to degrade DNA having free 5′ ends (dotted lines). (B) Hjr cleaved pUC(AT) products were incubated with buffer alone (-) (Lane 1), with lambda exonuclease (Lane 2), with T4 DNA ligase (Lane 3) or with T4 DNA ligase then lambda exonuclease (Lane 4) and separated by agarose gel electrophoresis.
SIRV2 Hjr interacts with host DNA binding proteins and SIRV2 coat protein (gp26)
To investigate what protein partners might function with Hjr, immunoprecipitation was used. MBP-Hjr was incubated with a cell-free extract of SIRV2-infected S. islandicus, containing protein, lipid, and nucleic acids. Components bound to the resolving enzyme were captured by magnetic anti-MBP beads and washed to reduce non-specific binding. Treatment of the washed beads with Factor X protease released Hjr and bound proteins. Several proteins in the 10–20 kD range and the 40–50 kD range were observed by SDS-PAGE (data not shown).
Proteins captured by MBP-Hjr immunoprecipitation were identified by mass spectrometry and are listed according to their presumed functional role (Table S3). MBP-Hjr forms complexes with proteins including the DNA binding proteins, Sso10b, Sso7d, Cren7 and Alba (Table S3). Interactions between MBP-Hjr and these DNA binding proteins could be a result of direct protein:protein interactions or may result from binding between MBP-Hjr and a nucleic acid that is in turn bound by a DNA binding protein. Most strikingly, an interaction between Hjr and the coat protein, SIRV2gp26 was identified with a high confidence value (Table 1). Further studies using purified proteins were implemented to verify this interaction with the SIRV2 coat protein.
SIRV2 Hjr and SIRV2 coat protein (gp26) interact in vitro
The putative direct interaction between purified MBP-Hjr and SIRV2gp26 was tested in vitro. As detected by SDS-PAGE, magnetic anti-MBP::MBP-Hjr beads pulled down SIRV2gp26 (Figure 5). Control reactions with MBP5 alone did not pull down SIRV2gp26 suggesting that the interaction is specific for Hjr rather than for the MBP domain of the fusion protein.
The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.
To address the molecular basis for substrate recognition and cleavage, we characterized the resolving enzyme from the hyperthermophilic virus, SIRV2. Hjr cleaves four-way junctions without cleaving other related DNA structures and contrasts with the bacteriophage resolving enzymes T4 endonuclease VII and T7 endonuclease I that recognize a wide range of DNA structures including four-way junctions, Y-structures, heteroduplex loops, single-strand overhangs, nicks, gaps, apyrimidinic sites, and base mismatches [32], [33], [34]. T4 endonuclease VII and T7 endonuclease I have large electropositive areas on the protein surface that bind to the four-way junction DNA backbone over a large area and are flexible and broad enough to allow binding and cleavage a variety of branched DNA substrates [35], [36]. Unlike bacteriophage resolving enzymes, Sso Hje, Sso Hjc, and SIRV2 Hjr substrate specificity is narrow and limited to X-shaped four-way junctions. The Sso Hje three-dimensional structure provides a model to explore elements that determine the narrow substrate range of Sso Hje and SIRV2 Hjr. Electropositive patches are arranged on the Sso Hje and Sso Hjc DNA binding surface in an X-shaped pattern [4], [18]. Presumably, this pattern of electropositive residues favors binding of X-stacked four-way junctions by Sso Hje and Hjc (and by extension, SIRV2 Hjr). Further structural studies of SIRV2 Hjr in complex with DNA will further reveal if positively charged surface amino acids are arranged in a pattern to bind X-stacked four-way junction substrates to confer substrate specificity.
SIRV2 Hjr has a unique strand cleavage preference that may reflect fundamental differences in Holliday junction recognition and cleavage. This preference is observed even while sharing sequence similarity with Sso Hje and Sso Hjc. Sso Hje is specific for cleavage of continuous strands in four-way junction J3 while Sso Hjc cleaves both continuous and exchange strands. SIRV2 Hjr presents a third cleavage pattern by nicking exchange strand pairs. Previous studies of Sso Hje and Hjc have suggested a structural basis to account for differences in resolving enzyme specificity and cleavage patterns (1). Sso Hje and Sso Hjc are both homodimers stabilized along the dimer interface by interactions between monomer amino acids (Figure 6A). Even thought the dimer interface is distal to the DNA binding region, Middleton and coworkers have argued that positioning of the dimers influences positioning of adjacent catalytic residues on the DNA. When Sso Hje and Sso Hjc three-dimensional structures are superimposed, the main chain Cα positioning is structurally well conserved with the largest differences observed at the dimer interface (Figure 6A). Specifically, an insertion of three large hydrophobic residues (M77, F78, M80) in a loop between helix α2 and beta strand βE of each Sso Hje monomer shifts the dimer conformation and may position catalytic residues for cleavage of continuous strands of a four-way junction. In contrast to Sso Hje, a relatively short Sso Hjc loop connecting helix α2 and beta strand βE may confer additional flexibility to allow cleavage of both exchange and continuous strands in a four-way junction. SIRV2 Hjr, like Sso Hje, also contains an insertion in the equivalent loop although the sequences diverge. Most notably, SIRV2 Hjr contains three cysteines (C77, C82, C84) in the insertion loop. These cysteines may form intra- or inter-monomer disulfide bonds to alter resolving enzyme conformation and thereby position adjacent catalytic residues to favor cleavage of exchange rather than continuous strands (Figure 6B). Therefore, variations in resolving enzyme dimer interfaces may uniquely position catalytic residues for pairwise nicking on either the continuous (Sso Hje), exchange (SIRV2 Hjr), or both (Sso Hjc).
(A) Sso Hje (accession: 1OB8) and Sso Hjc (accession: 1HH1) three-dimensional structures were aligned using MacPymol. Sso Hje monomers (red and blue) are visualized by a surface view to highlight the dimer arrangement. A domain formed by helix alpha-2 and beta strand beta-E form part of the dimer interface (arrow). In insertion in the loop between helix alpha-2 and beta strand beta-E (arrow) (Sso Hje: red and blue; Sso Hjc: green) shifts dimer conformation and may confer strand cleavage specificity. (B). The amino acid sequence of the Sso Hje, Sso Hjc, and SIRV2 Hjr dimerization interface helix alpha-2 and beta strand beta-E were aligned by Clustal W and the insertion loop is underlined. Amino acids conserved between Sso Hje, Sso Hjc, and SIRV2 Hjr are shaded in black and those conserved between Sso Hje and SIRV2 Hjrare shaded in gray.
The SIRV2 Hjr substrate cleavage specificity has the characteristics to function as a replication resolving enzyme in a poxvirus-like mode of viral DNA replication. In such a mechanism, linked genome concatamers are resolved via cleavage during replication [12]. SIRV2 Hjr action on such hairpin four-way junction concatamer DNA would produce linear DNA products with nicked hairpin termini, which in turn could serve as nicked templates for further replication. Therefore, the SIRV2 resolving enzyme likely plays a central role in the final stages of SIRV2 replication and functions in the general mechanism for separating concatamers during replication of linear viral genomes.
Finally, we have shown that in addition to cleaving four-way junctions, Hjr may have a role in viral assembly. It is tempting to speculate that an interaction between Hjr and coat protein could be the signal for assembly of the coat protein into a superfilament that surrounds the resolved SIRV2 genome, thereby forming the virion body. Known signals for viral packaging include DNA sequences, RNA structures, and non-structural proteins that recruit viral structural proteins to initiate assembly [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49]. For instance, vaccinia virus nucleates virion assembly via a transient interaction between a hairpin terminus-binding protein, I6, and the vaccinia virus coat protein [48], [49]. Therefore, like vaccinia virus I6 protein, the interaction between Hjr and coat protein could ensure that resolved, single genome copies rather than concatamers are packaged into virus particles. In addition, because both Hjr and coat protein sequences are highly conserved among rudiviruses, this nucleation model could also serve as a general strategy for virion packaging in related rudiviruses, Acidianus Rod-Shaped virus 1 (ARV1), Stygioglobus rod-shaped virus (SRV), and Sulfolobus islandicus rod-shaped virus 1 (SIRV1). Given the similarity between archaea and eukarya, it is also conceivable that similar encapsidation mechanisms may also exist in eukaryotic viruses.
Supporting Information
Figure S1.
Four-way junction DNA structures and sequences. (A) A poly(AT)20 cassette in pUC(AT) can form a four-way junction structure. (B) pEMM2 contains a four-way junction sequence at the junction of SIRV2 concatamers. These figures represents one of many conformations of possible mobile four-way junction structures. The four-way junction center may shift from what is represented. Gray regions are vector sequence.
Figure S2.
Mapping resolvase cleavage site on plasmid pEMM2. Plasmid pEMM2 contains a four-way junction sequence mimicking the four-way junction formed at SIRV2 concatamer junctions. pEMM2 (100 nM) was incubated with T7 endonuclease I (7.5 nM) (Lane 2), MBP-Hjr (20 nM) (Lane 3), or MBP-Hjr (2 nM) (Lane 4) at 37°C (T7 endonuclease I) or 55°C (MBP-Hjr) for 30 minutes. Then reaction products were then digested with 10 Units of XmnI at 37°C for one hour and separated by agarose gel electrophoresis. As a control (Lane 1), 1 µg pEMM2 was digested with 10 Units of XmnI and HindIII (proximal to the four-way junction) to generate a 921 bp and 1851 bp fragment. pEMM2 was digested with XmnI alone (Lane 5), XmnI and HindIII (proximal to the four-way junction). Plasmid not cleaved by T7 endonuclease I or SIRV2 Hjr and then cleaved with XmnI will generate a linear 2772 bp band. As expected, a double stranded break generated from T7 endonuclease I and MBP•SIRV2 Hjr cleavage mapped to the cruciform region of pEMM2 to generate a ∼875 bp and 1897 bp fragment (Lanes 2, 3, 4). (B) A plasmid map of pEMM2 illustrating HindIII, XmnI and cruciform cleavage site.
Figure S3.
Requirements for SIRV2 Hjr four-way junction cleavage. Hjr cleavage activity was assayed with DNA four-way junction J3 under a variety of reaction conditions to determine requirements for cleavage. Four-way junction J3 (FAM-labeled on strand b) (100 nM) and Hjr (20 nM) were incubated in reaction buffer at 55°C for 30 minutes. Reaction buffers varied pH (4–10), divalent cation (MgCl2, MnCl2, ZnSO4, CaCl2, CoCl2), concentration of NaCl (0–500 mM), NH4SO4 (0–200 mM), or MgCl2 (0–100 mM). Reactions were quenched by addition of 50% formamide and 5 mM EDTA. Reaction products were separated by 20% denaturing PAGE and fluorescence quantified using a GE Typhoon scanner. S and P represent substrate (34-mer) and product (18-mer) bands, respectively.
Table S1.
Oligonucleotides for SIRV2gp26, SIRV2 Hjr, and S. islandicus Hje gene synthesis.
Table S2.
Oligonucleotides for DNA substrates.
Table S3.
Proteins that interact with MBP-SIRV2 Hjr.
We are grateful to Jack Benner and Casey Madinger at the New England Biolabs for providing mass spectrometry analysis. We also thank Malcolm White at University of St. Andrews for providing Sulfolobus islandicus Hje and for helpful discussions; David Prangishvili for helpful discussions; Tom Evans, Fran Perler, and Lise Raleigh for critical review of this manuscript. We are also indebted to Don Comb for fostering a supportive research environment at New England Biolabs.
Author Contributions
Conceived and designed the experiments: AFG CG WEJ. Performed the experiments: AFG. Analyzed the data: AFG CG WEJ. Contributed reagents/materials/analysis tools: AFG CG. Wrote the paper: AFG CG WEJ.
- 1. Lilley DM, White MF (2001) The junction-resolving enzymes. Nat Rev Mol Cell Biol 2: 433–443.DM LilleyMF White2001The junction-resolving enzymes.Nat Rev Mol Cell Biol2433443
- 2. Déclais A-C, Lilley DMJ (2008) New insight into the recognition of branched DNA structure by junction-resolving enzymes. Current Opinion in Structural Biology 18: 86–95.A-C DéclaisDMJ Lilley2008New insight into the recognition of branched DNA structure by junction-resolving enzymes.Current Opinion in Structural Biology188695
- 3. White MF, Giraud-Panis MJ, Pohler JR, Lilley DM (1997) Recognition and manipulation of branched DNA structure by junction-resolving enzymes. J Mol Biol 269: 647–664.MF WhiteMJ Giraud-PanisJR PohlerDM Lilley1997Recognition and manipulation of branched DNA structure by junction-resolving enzymes.J Mol Biol269647664
- 4. Bond CS, Kvaratskhelia M, Richard D, White MF, Hunter WN (2001) Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus. Proceedings of the National Academy of Sciences 98: 5509–5514.CS BondM. KvaratskheliaD. RichardMF WhiteWN Hunter2001Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus.Proceedings of the National Academy of Sciences9855095514
- 5. Connolly B, Parsons CA, Benson FE, Dunderdale HJ, Sharples GJ, et al. (1991) Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. Proc Natl Acad Sci U S A 88: 6063–6067.B. ConnollyCA ParsonsFE BensonHJ DunderdaleGJ Sharples1991Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.Proc Natl Acad Sci U S A8860636067
- 6. Culyba MJ, Harrison JE, Hwang Y, Bushman FD (2006) DNA cleavage by the A22R resolvase of vaccinia virus. Virology 352: 466–476.MJ CulybaJE HarrisonY. HwangFD Bushman2006DNA cleavage by the A22R resolvase of vaccinia virus.Virology352466476
- 7. Ip SC, Rass U, Blanco MG, Flynn HR, Skehel JM, et al. (2008) Identification of Holliday junction resolvases from humans and yeast. Nature 456: 357–361.SC IpU. RassMG BlancoHR FlynnJM Skehel2008Identification of Holliday junction resolvases from humans and yeast.Nature456357361
- 8. Komori K, Sakae S, Shinagawa H, Morikawa K, Ishino Y (1999) A Holliday junction resolvase from Pyrococcus furiosus: Functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea. Proceedings of the National Academy of Sciences 96: 8873–8878.K. KomoriS. SakaeH. ShinagawaK. MorikawaY. Ishino1999A Holliday junction resolvase from Pyrococcus furiosus: Functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea.Proceedings of the National Academy of Sciences9688738878
- 9. Kvaratskhelia M, White MF (2000) Two holliday junction resolving enzymes in Sulfolobus solfataricus. Journal of Molecular Biology 297: 923–932.M. KvaratskheliaMF White2000Two holliday junction resolving enzymes in Sulfolobus solfataricus.Journal of Molecular Biology297923932
- 10. Muller B, Jones C, West SC (1990) T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein. Nucleic Acids Res 18: 5633–5636.B. MullerC. JonesSC West1990T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein.Nucleic Acids Res1856335636
- 11. Picksley SM, Parsons CA, Kemper B, West SC (1990) Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions. J Mol Biol 212: 723–735.SM PicksleyCA ParsonsB. KemperSC West1990Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions.J Mol Biol212723735
- 12. Blum H, Zillig W, Mallok S, Domdey H, Prangishvili D (2001) The genome of the archaeal virus SIRV1 has features in common with genomes of eukaryal viruses. Virology 281: 6–9.H. BlumW. ZilligS. MallokH. DomdeyD. Prangishvili2001The genome of the archaeal virus SIRV1 has features in common with genomes of eukaryal viruses.Virology28169
- 13. Birkenbihl RP, Neef K, Prangishvili D, Kemper B (2001) Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2. Journal of Molecular Biology 309: 1067–1076.RP BirkenbihlK. NeefD. PrangishviliB. Kemper2001Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2.Journal of Molecular Biology30910671076
- 14. Neef K, Birkenbihl RP, Kemper B (2002) Holliday junction-resolving enzymes from eight hyperthermophilic archaea differ in reactions with cruciform DNA. Extremophiles 6: 359–367.K. NeefRP BirkenbihlB. Kemper2002Holliday junction-resolving enzymes from eight hyperthermophilic archaea differ in reactions with cruciform DNA.Extremophiles6359367
- 15. Dorazi R, Parker JL, White MF (2006) PCNA Activates the Holliday Junction Endonuclease Hjc. Journal of Molecular Biology 364: 243–247.R. DoraziJL ParkerMF White2006PCNA Activates the Holliday Junction Endonuclease Hjc.Journal of Molecular Biology364243247
- 16. Kvaratskhelia M, Wardleworth BN, Norman DG, White MF (2000) A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc. J Biol Chem 275: 25540–25546.M. KvaratskheliaBN WardleworthDG NormanMF White2000A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc.J Biol Chem2752554025546
- 17. Kvaratskhelia M, Wardleworth BN, White MF (2001) Multiple Holliday junction resolving enzyme activities in the Crenarchaeota and Euryarchaeota. FEBS Letters 491: 243–246.M. KvaratskheliaBN WardleworthMF White2001Multiple Holliday junction resolving enzyme activities in the Crenarchaeota and Euryarchaeota.FEBS Letters491243246
- 18. Middleton CL, Parker JL, Richard DJ, White MF, Bond CS (2004) Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje. Nucl Acids Res 32: 5442–5451.CL MiddletonJL ParkerDJ RichardMF WhiteCS Bond2004Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje.Nucl Acids Res3254425451
- 19. Parker JL, White MF (2005) The Endonuclease Hje Catalyses Rapid, Multiple Turnover Resolution of Holliday junctions. Journal of Molecular Biology 350: 1–6.JL ParkerMF White2005The Endonuclease Hje Catalyses Rapid, Multiple Turnover Resolution of Holliday junctions.Journal of Molecular Biology35016
- 20. Czar MJ, Anderson JC, Bader JS, Peccoud J (2009) Gene synthesis demystified. Trends Biotechnol 27: 63–72.MJ CzarJC AndersonJS BaderJ. Peccoud2009Gene synthesis demystified.Trends Biotechnol276372
- 21. Reno ML, Held NL, Fields CJ, Burke PV, Whitaker RJ (2009) Biogeography of the Sulfolobus islandicus pan-genome. Proc Natl Acad Sci U S A 106: 8605–8610.ML RenoNL HeldCJ FieldsPV BurkeRJ Whitaker2009Biogeography of the Sulfolobus islandicus pan-genome.Proc Natl Acad Sci U S A10686058610
- 22. Eichman BF, Vargason JM, Mooers BH, Ho PS (2000) The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions. Proc Natl Acad Sci U S A 97: 3971–3976.BF EichmanJM VargasonBH MooersPS Ho2000The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions.Proc Natl Acad Sci U S A9739713976
- 23. Guan C, Kumar S (2005) A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease. Nucleic Acids Res 33: 6225–6234.C. GuanS. Kumar2005A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease.Nucleic Acids Res3362256234
- 24. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.SF AltschulW. GishW. MillerEW MyersDJ Lipman1990Basic local alignment search tool.J Mol Biol215403410
- 25. Culyba MJ, Minkah N, Hwang Y, Benhamou OM, Bushman FD (2007) DNA branch nuclease activity of vaccinia A22 resolvase. J Biol Chem 282: 34644–34652.MJ CulybaN. MinkahY. HwangOM BenhamouFD Bushman2007DNA branch nuclease activity of vaccinia A22 resolvase.J Biol Chem2823464434652
- 26. Dickie P, McFadden G, Morgan AR (1987) The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I. J Biol Chem 262: 14826–14836.P. DickieG. McFaddenAR Morgan1987The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I.J Biol Chem2621482614836
- 27. Culyba MJ, Hwang Y, Minkah N, Bushman FD (2009) DNA binding and cleavage by the fowlpox virus resolvase. J Biol Chem 284: 1190–1201.MJ CulybaY. HwangN. MinkahFD Bushman2009DNA binding and cleavage by the fowlpox virus resolvase.J Biol Chem28411901201
- 28. Komori K, Sakae S, Fujikane R, Morikawa K, Shinagawa H, et al. (2000) Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus. Nucl Acids Res 28: 4544–4551.K. KomoriS. SakaeR. FujikaneK. MorikawaH. Shinagawa2000Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus.Nucl Acids Res2845444551
- 29. Murchie AI, Portugal J, Lilley DM (1991) Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange. EMBO J 10: 713–718.AI MurchieJ. PortugalDM Lilley1991Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange.EMBO J10713718
- 30. Murchie AI, Clegg RM, von Kitzing E, Duckett DR, Diekmann S, et al. (1989) Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature 341: 763–766.AI MurchieRM CleggE. von KitzingDR DuckettS. Diekmann1989Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules.Nature341763766
- 31. Duckett DR, Murchie AIH, Diekmann S, von Kitzing E, Kemper B, et al. (1988) The structure of the holliday junction, and its resolution. Cell 55: 79–89.DR DuckettAIH MurchieS. DiekmannE. von KitzingB. Kemper1988The structure of the holliday junction, and its resolution.Cell557989
- 32.
Kemper B (1997) Branched DNA resolving enzymes (X-solvases). In: Nickoloff JA, Hoekstra M, editors. Ottowa: Humana Press. pp. 179–204.B. Kemper1997Branched DNA resolving enzymes (X-solvases).JA NickoloffM. HoekstraOttowaHumana Press179204
- 33. Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, et al. (2003) Bacteriophage T4 genome. Microbiol Mol Biol Rev 67: 86–156, table of contents.ES MillerE. KutterG. MosigF. ArisakaT. Kunisawa2003Bacteriophage T4 genome.Microbiol Mol Biol Rev6786156, table of contents
- 34. Kemper B, Brown DT (1976) Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA. J Virol 18: 1000–1015.B. KemperDT Brown1976Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA.J Virol1810001015
- 35. Hadden JM, Declais AC, Carr SB, Lilley DM, Phillips SE (2007) The structural basis of Holliday junction resolution by T7 endonuclease I. Nature 449: 621–624.JM HaddenAC DeclaisSB CarrDM LilleySE Phillips2007The structural basis of Holliday junction resolution by T7 endonuclease I.Nature449621624
- 36. Biertumpfel C, Yang W, Suck D (2007) Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature 449: 616–620.C. BiertumpfelW. YangD. Suck2007Crystal structure of T4 endonuclease VII resolving a Holliday junction.Nature449616620
- 37. Khromykh AA, Varnavski AN, Sedlak PL, Westaway EG (2001) Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus. J Virol 75: 4633–4640.AA KhromykhAN VarnavskiPL SedlakEG Westaway2001Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus.J Virol7546334640
- 38. Patkar CG, Kuhn RJ (2008) Yellow Fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functions. J Virol 82: 3342–3352.CG PatkarRJ Kuhn2008Yellow Fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functions.J Virol8233423352
- 39. Annamalai P, Rofail F, Demason DA, Rao AL (2008) Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration. J Virol 82: 1484–1495.P. AnnamalaiF. RofailDA DemasonAL Rao2008Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.J Virol8214841495
- 40.
Baltimore D (1969) The replication of picornaviruses. In: Levy HB, editor. The biochemistry of viruses. New York: Marcel Dekker, Inc. pp. 101–176.D. Baltimore1969The replication of picornaviruses.HB LevyThe biochemistry of virusesNew YorkMarcel Dekker, Inc101176
- 41. Black LW (1989) DNA packaging in dsDNA bacteriophages. Annu Rev Microbiol 43: 267–292.LW Black1989DNA packaging in dsDNA bacteriophages.Annu Rev Microbiol43267292
- 42. Koerner JF, Snustad DP (1979) Shutoff of host macromolecular synthesis after T-even bacteriophage infection. Microbiol Rev 43: 199–223.JF KoernerDP Snustad1979Shutoff of host macromolecular synthesis after T-even bacteriophage infection.Microbiol Rev43199223
- 43. Kruger DH, Schroeder C (1981) Bacteriophage T3 and bacteriophage T7 virus-host cell interactions. Microbiol Rev 45: 9–51.DH KrugerC. Schroeder1981Bacteriophage T3 and bacteriophage T7 virus-host cell interactions.Microbiol Rev45951
- 44. Marvin DA (1998) Filamentous phage structure, infection and assembly. Curr Opin Struct Biol 8: 150–158.DA Marvin1998Filamentous phage structure, infection and assembly.Curr Opin Struct Biol8150158
- 45. McBride MS, Schwartz MD, Panganiban AT (1997) Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J Virol 71: 4544–4554.MS McBrideMD SchwartzAT Panganiban1997Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation.J Virol7145444554
- 46. Mansky LM, Wisniewski RM (1998) The bovine leukemia virus encapsidation signal is composed of RNA secondary structures. J Virol 72: 3196–3204.LM ManskyRM Wisniewski1998The bovine leukemia virus encapsidation signal is composed of RNA secondary structures.J Virol7231963204
- 47. Zeffman A, Hassard S, Varani G, Lever A (2000) The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein. J Mol Biol 297: 877–893.A. ZeffmanS. HassardG. VaraniA. Lever2000The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein.J Mol Biol297877893
- 48. Grubisha O, Traktman P (2003) Genetic analysis of the vaccinia virus I6 telomere-binding protein uncovers a key role in genome encapsidation. J Virol 77: 10929–10942.O. GrubishaP. Traktman2003Genetic analysis of the vaccinia virus I6 telomere-binding protein uncovers a key role in genome encapsidation.J Virol771092910942
- 49. Chung CS, Chen CH, Ho MY, Huang CY, Liao CL, et al. (2006) Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles. J Virol 80: 2127–2140.CS ChungCH ChenMY HoCY HuangCL Liao2006Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles.J Virol8021272140


text:
There was good reason to consider Walsh to be virtually a lock to make field goals. After all, of his 38 attempts this season, he only missed three and was perfect from 50 yards and beyond (10-for-10). The first points of Sunday's 37-34 win over Green Bay that put the Vikings in the playoffs came on a 54-yard field goal from Walsh. With 24 seconds to play, it appeared as though the team was going to need another long distance call from Walsh to win the game (until Adrian Peterson performed his own style of magic once again).
Even Walsh seemed a little surprised by the Vikings' tactics in the final two minutes.
"I understood the thinking on it – just give the ball to A.P. and let him get as many yards as he could get," Walsh said. "But it seemed like once we got inside the 40-yard line, we weren't in a hurry. I guess they figured we had got to a distance that I could make it from."
The fact that Walsh has made every kick he has tried from 50 yards and beyond was a testament to his calm under pressure and his ability to come through when the odds tell you field goals from that distance are about a 50-50 proposition.
"He just doesn't get rattled," long snapper Cullen Loeffler said. "We were talking about it on the sidelines in that final drive and, when we started running the ball and letting the clock wind down, I told him it looked like it was going to be another long one to win the game. He just shrugged as if it was no big deal. Nothing seems to get to him."
His job was made markedly easier when Peterson broke off a 26-yard run to the Packers' 11-yard line, turning what would have been an attempt of between 54 and 55 yards into a 29-yard chip shot.
"You've got to love Adrian," Walsh said. "I was confident I could make the field goal from where the ball was before the play, but he made things so much easier with what he did on that last play."
As the Vikings head to the playoffs for a rematch with Green Bay next Saturday night, they do so knowing that Walsh may have to kick another field goal to put them over the top for a playoff win. While the prospect of kicking at temperatures near zero doesn't thrill Walsh, he's ready for the challenge.
"You never know how a game is going to turn," Walsh said. "Some games you attempt four or five field goals. In other games, you're just kicking extra points. I'd love to kick a bunch of extra points, but, if we need a field goal to try to win another game, I'll be ready."
"It's a bittersweet feeling inside," Peterson said. "The first thing that came to mind when I heard that I was 9 yards short was, 'It is what it is. It wasn't meant to happen.' Not to say that it doesn't hurt, it does."
"I'll be fine," Winfield said. "All we have do is get the swelling out of it and I'll be good to go. It hurts now, but you play with pain in this league."
"I'm geeked up every game," Griffen said. "They always have to tell me to calm down and hold my composure. This was a huge game for us and I was running full speed all day long."
John Holler has been writing about the Vikings for more than a decade for Viking Update. Follow Viking Update on Twitter and discuss this story on our subscriber message board.

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text: Posted September 22, 2010 In breaking news this morning,Associated Press reported that NASCAR was taking a closer look at Clint Bowyer's winning #33 car that won Sunday's race. If NASCAR decides to penalize Bowyer points for an infraction, then this could seriously hurt him in the championship chase. http://www.rr.com/sports/topic/article/rr/9003/21976327/AP_Source_NASCAR_taking_closer_look_at_car_No_33 1 person likes this Share this post Link to post Share on other sites


text:
1966 Ford Mustang Convertible
1964½ Ford Mustang
1973 Ford Mustang Mach 1
1986 Ford Mustang SVO
1988 Ford Mustang GT 5.0 Convertible
In the run up to the big 50th anniversary of the Mustang, prices of the classic first version of the Mustang, the 1964½ (technically a 1965 model) through 1966, have been moving up. The best convertibles and fastbacks with the highest horsepower engines can bring well over $65,000 and there has been renewed interest in vintage Mustangs of all years. All isn't lost however for the Mustang collector on a budget. Here are five of our favorites that you can still afford:
In a world where the phrase "there's no replacement for displacement" is thrown around quite a bit, we tend to forget about some of the truly great small-block … More
Each January, the Scottsdale based Barrett-Jackson Classic Car Auction sets the stage for a new year of buying and selling collector vehicles – many regard it a … More
In 1964, more than 92,000 Ford Mustang Hardtops and close to 29,000 Ford Mustang Convertibles reached dealer showrooms, much to the appreciation and pleasure of … More
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|Home > Entertainment > Film > Article
|Saturday March 9, 2002
"In reflecting contemporary community standards across all classifiable elements of the film, the supernatural themes and language could not currently be accommodated at the G level of classification," OFLC director Des Clark said.
"It is understandable that attitudes shift over a 20-year period. This results in some films receiving different classifications when classified now."
But UIP managing director Mike Selwyn described the decision as "daft" and said Australia's censors were "overlooking the big picture".
"I think it's strange," Mr Selwyn told AAP.
"What's happened with ET is that Spielberg has put the film back together digitally, added in new footage, a digital soundtrack and it's still the same film that everybody loves but it's just got better.
"We put it in for classification and they decided that after it's been a G for 20 years they have to turn it into a PG, which is a little bit odd."
He said the decision was made even more surprising by the fact that Spielberg had re-examined elements of the film and toned down parts of it.
Guns carried by agents in the film were subsequently removed by Spielberg and replaced by walkie talkies and there was nothing in the new footage to impact the language and themes of the original film, Mr Selwyn said.
"What they've said is that it contains coarse language and a supernatural theme," he said.
"Well, I don't think it's any surprise to anybody that a film about aliens could have a supernatural theme to it.
"And the daftest thing is that all of the language was in the original version that was classified G, 20 years ago."
Mr Selwyn said UIP would appeal the decision and hoped commonsense would prevail so that ET regained its G rating.
The OFLC confirmed the Classification Review Board had received an application for review of the decision from UIP.
The Review Board meets to hear applications for review of decisions of the Classification Board.
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In this section
Cruise to snub Oscars to avoid Kidman embarrassment
A Beautiful Mind debuts at number one
Glimpses of Howard's good intentions
Waking life is but a dream
Enter the cinema at your own risk
Bettany noir
Hollywood takes aim at the pirates
Film's most inside outsider
Short cut to fame
A digital head-trip
Second time a Virgin?
Our fragile Oscar treasure
Mind fails to equate
Following the footsteps of women leads full circle
The Russ and Ron show rolls on
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Littleton, COUSA
June 25, 2012
No answer yet
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The Colorado Professional Videographers Association (www.CoPVA.org) consists of a group of videographers promoting the goodwill and advancement of videography in the Colorado area. Our mission is to provide in-depth knowledge to those in our field through monthly seminars, to provide networking opportunities and to share and receive new ideas, techniques and technology covering the many of fields of videography. In a word, at every meeting we hope you will learn, be inspired and meet someone new. For more information about our group go to www.copva.org.
10% off any video equipment rental simply by mentioning CoPVA
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Red Striped Vase
Bright fun colors reminds me of the 1970's. Tear drop shape makes it a little different from the norm. Can see this as a Valentine's Day gift. Funky, mod rooms will need this piece of art to add character to the room.
Echoing the rustic look of an apple orchard, these decorative faux fruits add natural warmth to a tabletop or console.Tags: Valentine's Day decorAdded 6 Years Ago from Pottery Barn
Sometimes the Kitchen is the most romantic place in your house. Why? Food is the key to any man's heart. Spice it up a bit with these Valentine Dishtowels.Tags: cute, Valentines, hostess gifts, kitchen decor, Pretty, kitchen items, Valentine's Day Gifts for Women, Valentine's Day for Anyone!, Kitchen DishtowelsAdded 10 Years Ago from Crate and Barrel


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This property is no longer available.Search for Available Property.
5.67 acres in Cherokee County, Georgia
Gorgeous Tract of land. Fairly Level... Beautiful trees and a creek runs through the property. Come build your dream home.
Take 108North and turn left on Puckett rd. Then right on Pine Bark Trail. Look for sign on the left.
Tax Data (Show Tax Data)
Tax data is not available for this listing.
Contact the Seller for more information.
Other Land for Sale in Cherokee County
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NOTE: The information contained herein is provided as general information only and has been obtained from sources deemed reliable. It is provided without any guaranty, warranty or representation, expressed or implied, made by Lands of America, or any related entity, as to the accuracy or completeness of the information. The information is presented subject to errors, omissions, change of price or conditions, prior sale or withdrawal without notice. Prospective purchasers should make their own investigations, projections and conclusions concerning the information.


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Dr. Kiarash N. Michel's Credentials
Accepting New Patients
Yes, this doctor is accepting new patients
Dr. Kia Michel is a renown and highly sought after Urologist in Los Angeles. His expertise is sought after by patients nationally and internationally. He is one of the few Urologists who is both an acclaimed cancer and robotic surgeon. Dr. Michel is a compassionate Urologist who has dedicated his career to providing state of the art care for his patients with a warm, and nurturing touch. He is highly respected by his peers and serves as an expert consultant to his colleagues. Dr. Michel is one of the founding members of the reputable Comprehensive Urology Medical Group, located in the Cedars-Sinai Medical Towers in Los Angeles, California. Comprehensive Urology Medical Group provides a personalized approach for helping patients with Urologic conditions. Dr. Michel has an illustrious educational path. Dr. Michel was a Presidential Scholar for four years at Whitman College. He subsequently attended the University of Washington School of Medicine, where he graduated with Honors. After completing his medical training, Dr. Michel was recruited by UCLA Department of Urology where he completed his residency. While at UCLA, Dr. Michel was recognized as a national Pfizer Scholar. After completing his residency, Dr. Michel completed additional training and developed further expertise in urologic cancer surgery. During this time, Dr. Michel was actively involved with research in the field of Urologic Cancers. His efforts have been recognized by the National Cancer Institute, the National Institute of Health, and the American Urological Association. Dr. Michel has expertise in both traditional ("open") surgical techniques as well as laparoscopic, minimally invasive and robotic surgical approaches. He provides the latest techniques and approaches for the treatment of urologic conditions.
- Farsi
- University Of Washington School Of Medicine (Grad. 1993)
- University of California, Los Angeles
Other Specialties
Areas of Expertise
- Prostate cancer
- Bladder cancer
- Kidney cancer
- Testicular cancer
- Benign prostatic hyperplasia (BPH)
- Urinary incontinence
- Erectile dysfunction
- Kidney stones
- Nephrectomy (kidney removal)
- Penile implants
- Transurethral resection of the prostate (TURP)
Awards & Recognitions
Publications & Research


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Enjoy the ultimate VIP treatment at Amalie Arena. Full season and 21-pack customers are welcomed with valet parking and a private entrance for hockey games, concerts, events, family shows and football. Be treated like royalty with complimentary food and beverages (including cocktails!) with your tickets. Never miss a moment of the action. The Lexus Lounge is adjacent to the locker rooms, with seats ten feet from the ice. Socialize with the who's who of Tampa Bay and be entertained in VIP style.
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Back to School, Back to Sleep
Fixing your children's sleep problems may improve their grades and their behavior.
By Michael Breus
Reviewed By Stuart Meyers
The new school year is upon us. From bedtime battles to the misery of morning call, summertime sleep habits die hard. Late summer nights combined with early school start times, and the stresses of just being a kid, deprive our children of essential sleep. And sleep deprivation often wreaks havoc with health, academic performance, and behavior. It is an unrecognized epidemic.
From elementary school through high school and beyond, a great many of our children are chronically sleep-deprived. With more than two-thirds of elementary school-age children having some kind of sleep problem and most adolescents not getting enough sleep, many will struggle to meet the barrage of new challenges, demands, and emotions of a new school year.
It is not widely recognized and appreciated just how pervasive and critical quality sleep is for brain development and how it directly influences daytime functioning, performance, mood, and behavior. When was the last time your doctor asked about your child's sleep? Parents wouldn't think of letting their child skip meals or run into a busy street, but staying up late is very often of little concern. It shouldn't be.
Sleep Affects How Your Child Thinks, Feels, and Functions
More and more research studies demonstrate that daytime sleepiness from chronic sleep deprivation and poor-quality sleep has significant affects on daytime behavior as well as concentration, attention, and mood. Even 20 fewer minutes of needed sleep may significantly affect behavior in many areas. One study showed that those students with C's, D's and F's got about 25 fewer minutes op sleep and went to bed an average of 40 minutes later than A and B students. The pediatric research findings are startling and alarming:
From Elementary to High School, Sleep Problems Are Pervasive and Widespread
You may be surprised to learn how prevalent sleep problems are. In studies of elementary school-age children, nearly 40% showed some kind of sleep problem, and 10% had daytime sleepiness. Up to half of adolescents reported at least occasional difficulty falling or staying asleep, with up to 13% experiencing chronic and severe insomnia. Clearly, the sleep time most teenagers get is insufficient: the average is under 7 ½ hours, with only 15% sleeping 8 ½ hours or more on school nights and more than 25% typically sleeping 6 ½ hours or less.
Results from the National Sleep Foundation's 2004 Sleepiness in America poll may (or may not!) surprise you:
Does My Child Have a Sleep Problem? Awareness Is Key
Given the prevalence and enormous impact of sleep problems on daytime functioning, we should all regularly look at our own children to see if they are getting the sound sleep they deserve. For some it may be obvious, but for most of us it requires some education, investigation, and a keen, watchful eye. This is because few of us really know what normal, healthy sleep should be, plus there is a great deal of individual variation among children and at different ages. Sleep deprivation is also difficult to detect because sleep problems are masters of disguise, often masquerading in myriad manifestations. Consider that:
What Can I Do? Think Sleep!
With so many hidden faces of sleep deprivation, you must be aware and "think sleep." As a parent, if you do not recognize the problem, it may well go unrecognized. You will also likely implement any remedies required. Early intervention is important, given the consequences and that children do not "grow out of" sleep problems; rather, the sleep problems of childhood tend to persist into adulthood.
Talk with your child. Just taking the time to ask some of the right questions can go a long way. If there is a problem, discuss it and make a plan agreeable to all. Include detailed targeted behavior changes and rewards.
With your child in school during the day and with after-school activities and part-time jobs for many, it is critical to talk with teachers, coaches, bosses, etc. to see if your child is exhibiting any signs of sleep deprivation. This may involve you taking some time to educate the educators.
Teach your child good sleep habits, also known as "sleep hygiene." Establish sleep-healthy bedtimes, bedtime routines, habits, and diets. Identify and reduce as much daytime stress as possible. Limit TV and other "screen time" (computers, video games), especially at bedtime, and do not put a TV in your child's bedroom; children with a TV in their room tend to go to bed later and get less sleep than those without, and those kids who get less sleep are more likely to spend two or more hours watching TV.
As in everything in life, do your best to be consistent and ask for help. Do not hesitate to call your doctor or sleep specialist. It's too important not to.


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Tuesday, February 28, 2012 4:10 pm
By Polyana da Costa · Bankrate.com
Our Spocs know "the financial situation of the borrowers better than their spouses," says Mark Atencio, senior vice president for mortgage servicer Green Tree Servicing. "They are that close and interacting that much with the borrower." Do you feel that close to your Spoc? I guess I need to tell you who Spoc is. A


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Loaner Program :
Our services include a loaner program in which we provide laptops, projectors, camcorders, a/v cables, and more for faculty use on a daily basis. All loans are subject to availability.
The AVT loaner program currently has laptops, projectors, camcorders, and VGA cables available for faculty and staff use. We are proud to say that our loaner program is growing and as more equipment becomes available, it will be added to our list.
All equipment is loaned on a first-come first-serve basis. Equipment must be returned the same day it is picked up. No exceptions! Based upon need, each device has a specified checkout time-limit.
You have to come toto loan the equipment.
Faculty Training :
AVT also provides faculty training for utilizing equipped classrooms. Training is provided either on a one-on-one basis (referred to as "lite" training) or in a group setting. Lite training is scheduled through our office with at least 24 hour notice and based on availability of technicians. Group training announcements are emailed to all faculty and staff at least two weeks prior to scheduled date.
Repairing of A/V equipment :
AVT is also responsible for repairing all A/V equipment in the classrooms (with the exception of anything PC related). Any A/V equipment malfunction should be reported to our office immediately.


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- Internships /
- Fashion /
- Social Media Fashion/Wellness Start Up
Similar Internships
Media Intern (Film/Video/Photo)X by Gottex
Event CoordinatorNVEI Fashion Agency
Assistant StylistSociety Row
Social Media StylistSociety Row
PR/Social Media InternRebecca Minkoff
Small, fun, Fashion Company seeks an Office InternLori Goldstein Ltd.
Fashion Marketing & Sales InternshipLana Unlimited
Photographer/Social Media Intern for Mens Apparel companyNew Standard Edition
Social Media Fashion/Wellness Start UpBookmark This
Sara Yo Healing Jewelry
Posted: February 21 2016
Company Location: Tiverton, RI
Application Deadline: No Deadline
Position: Part-time, Unpaid
Always be on the alert for potentially fraudulent job postings online - never send money to a potential employer.
DescriptionWe started a business a year and a half ago called Sara Yo Spirit Jewels to help people overcome obstacles in their lives. The foundation of the business is based on two factors. One is a healing messaging service based on ancient Chinese Tsao philosophy/Five-Element energy and the other is jewelry comprised of five element healing properties. This five-element energy has been used in China for treating diseases and mental wellbeing for over 2,000 years and recently this philosophy has expanded to the western world and is getting more and more popular like yoga. Management consultant companies have started to use this five-element philosophy to guide executives to more effective day-to- day operations and to help them grow in their personal lives. This jewelry is quite unique as each piece is exclusively designed by Hannah and looks like no other jewelry on the market today. The foundation of Sara Yo jewelry involves a rare stone Hannah discovered near her hometown in Southeast China. The stone is made from a mineral called Kaolin, which formed under ground for millions of years. The jewelry is not only gorgeous but also functional. Under our guidance with the healing service we provide, women use their Sara Yo healing jewelry as a meditation tool to improve in areas where they lack certain types of energy. We created our own unique healing service, which is our intellectual property, to include a computerized five-element energy test system. Based on this system, we generate customized reports letting our customers know what types of energy they are lacking and then suggest pieces that relate to the areas of energy where they are deficient. When customers purchase items, we send them weekly healing messages to guide them on using Sara Yo pieces for meditation.
Since starting the business one and a half years ago, we have sold hundreds of pieces. Feedback from our customers is very encouraging, as they feel much better about themselves after wearing Sara Yo Spirit Jewels and many of them say they cannot live without their Sara Yo pieces. Because of the positive results we have seen, Hannah has left her lucrative medical career as a doctor to devote full time efforts to this business. We have learned that when women hear about the story, they get excited and purchase our items and come back for more. Why? Because they need different shapes of stones, as each stone shape represents a different type of energy to support their energy needs. Therefore, having an ongoing forum to tell the Sara Yo story is key to our success.
Hannah's background as medical doctor and Harvard trained scientist brings a truly unique competitive advantage over other jewelry companies. The story of her career change from medical to fashion/natural healing via Sara Yo, has attracted lots of media attention. Sara Yo stories have been featured on ABC TV, Fox TV, CW TV and many fashion and health magazines. For example, the well-respected health magazine, Natural Awak
ResponsibilitiesOur intern will be involved in:
*Researching social media opportunities in the fashion field
*Researching social media opportunities in the wellbeing field
*Uncovering social media programs relating to branding our start up business
*Creating a social media strategy for Sara Yo Healing Jewelry
*Helping Sara Yo attract new customers via a robust social media program
RequirementsWe are interested in an intern who possesses strong passion and abilities in the area of social media.
SkillsRequired - Social Media Marketing, Social Media Advertising, Social Media Consulting, Marketing for Small Business
Preferred - Energy Healing, Fashion Marketing, Social Media Development


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Midbrain is actually a small region in an important human brain which mainly assists while the relay hub for the various actions love auditory, visual and electric motor systems. Consequently, the Midbrain Service process can be in actuality a method to the Midbrain Service, which focuses on advancement of a child's learning functionality and increases his understanding in aesthetic, auditory and many different approaches.

How to activate Midbrain?

Most of the parents are curious to know how to activate Midbrain? Debunking 'midbrain service' of kids There can be MindTech's Outstanding Midbrain Account activation Teaching plan which is normally targeted on the tremendous rate learning. There happen to be several approaches for the Midbrain schooling, which all round raises the learning ability of your children and will support them in the change for better as the excellent kid forcing behind all the several other children of the exact get older. Mid-Brain Account activation Schooling can be a approach that is employed to activate the 'Mid-Brain'. The goal is to unleash the child probable through the service of 'Mid-Brain'. 'Mid-Brain' can be the bridge for relating up and levelling the capabilities of the kept and right thought process. Triggering it would enable both still left and right human brain to be utilised all in one go. You may want to call it as "Blind Fold Analyzing", or "Blind Fold Investigation", or "Blind Fold Viewing" etc., because the people will be blind folded during the workshop.

MindTech Midbrain Teaching Locations found in India and also Overseas.

There are several centers around several parts of India, which are focused in providing Midbrain Activation training. This is definitely your 1st step towards a Wonderful Imagination Technician Business Prospect. We happen to be excited at the potential of having you as a component of Brain Technician family group, which is usually most effective developing MidBrain Initial enterprise. We will be having more than 400 franchisees all over India and international. We give you the best extreme value in the industry with teaching, certification and support for growing faster. Consequently, what people receive is definitely the workout applications in either their individual cities or near their home.


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Add contemporary sophistication to any bedroom with a classic white upholstered bed
YUP's eco-friendly upholstered bed features recycled corrugated fibres and repurposed foam backing. It's available in pleather and microsuede. From $349 through www.yupinc.com.
IKEA 's Vanvik bed frame is an inexpensive take on the upholstered look, featuring a removable, machine-washable cover for easy maintenance. From $399 through www.ikea.ca.
EQ3's contemporary Dune bed features a sleek, modern profile and fully upholstered headboard, footboard and rails. From $1,199 through www.eq3.com


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The following products and vehicles were recalled by the Consumer Product Safety Commission or the National Highway Traffic Safety Administration. Unless otherwise indicated, discontinue use of the products immediately and return them to the store where purchased for a refund. For more information about the products, call the manufacturer or CPSC's toll-free hotline, 800-638-2772. Only some cars or trucks recalled are affected. Contact a dealer for your model to see if it is included in the recall. The dealer will tell you what to do.
Product • Air Mister, sold at Target stores
Problem • The Air Mister can shatter during use, posing an injury hazard. Consumers can contact Target Guest Relations at 1-800-440-0680 from 6 a.m. to 7 p.m., Monday through Friday, or online at www.target.com. Click on "Product Recalls" at the bottom of the page, and then select "Sports, Outdoor & Recreation."
Vehicle • Polaris Global Electric Motorcars, 2010-2013
Problem • Polaris is recalling certain model year 2010-2013 Global Electric Motorcars manufactured from Jan. 14, 2010, through Nov. 23, 2012. The parking brake has mechanical components that may wear prematurely and prevent the brake from engaging. Polaris recall numbers are L-13-01 and L-13-02. For assistance, call Polaris toll free at 1-888-871-4367. Have your vehicle's VIN handy. Owners may also contact the National Highway Traffic Safety Administration's Vehicle Safety Hotline at 888-327-4236 (TTY: 800-424-9153), or go to www.safercar.gov.


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Who would you cast for Martian reality TV?
Updated 7:49 pm, Thursday, May 9, 2013
So far, 78,000 people have signed up for a one-way ticket to Mars aboard whatever spacecraft the marketing firm Mars One finds for a series of trips to the Red Planet starting in 2023.
… and flying to the Red Planet is only part of what makes the trip an interesting idea. Think "Jersey Shore," only 140 million miles away (on average).
Mars One has a 10-year plan for finding, selecting and training the astronauts and will broadcast the entire affair in reality-TV fashion.
When they land, the first four members of the Mars colony – two men and two women? Same-sex partners? All women? All men? – will be joined every two years by new colonists until 40 people are encamped on the planet.
Mars One founder Bas Lansdorp predicts the mission will cost $6 billion, Mashable writes. Mars One, which maintains that it's a non-profit entity, won't rely on federal funding for support. Its business plan hinges on advertising in the reality TV setting.
If you could choose the 40 people who will spend the rest of their lives on the Red Planet – while being filmed 24/7/365 – who would you send?
Check out the above gallery to see our choices.


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I recently had an exchange in which someone mentioned leaving the church. Who hasn't? He quit participating some time back, and he's not alone.There are any number of reasons people walk out and don't return. There are those who wake up one day and decide the whole enterprise is false. But most seem to leave because the church has failed to measure up in some way. They leave because they are disillusioned.You know the triggers: Community life is lacking. A fellow parishioner said … [Read more...]
Disillusioned by the church
April 30, 2013 by 5 Comments


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I had a wonderful birthday! After leaving Ben at his favourite kennels, I hopped on the train to Bristol to spend the weekend being spoiled by all my family. I was taken out for lunch (twice!), went shopping and saw the Hobbit - in a VIP seat with chocolate fudge icecream! I really enjoyed the … [Read more...]
Chillington prepares … free Scrappy Union Jack pattern
Chillington, where I live, is a small, sleepy village nestling in the lush green south Devon countryside, just a couple of miles from the sea. It is the sort of place where nothing ever seems to happen - until now!!! The Olympic torch will be carried through OUR village on its journey around … [Read more...]


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11/10/99- Updated 09:45 AM ET
Gore seeks black support
MEMPHIS, Tenn. (AP) - In recent weeks, Vice President Al Gore has tried to shed his
scripted speeches and get away from the microphone - to appear less wooden and more
A slew of pastors had warmed the crowd to a near frenzy of singing and praying in the hour and a half before Gore took the stage at Mason Temple. That was where the Rev. Martin Luther King gave his prophetic ''Mountaintop'' speech before an assassin took his life, and where President Clinton gave an emotional speech six years ago.
During the nearly hourlong speech, Gore quoted King's speech, told jokes and biblical stories, and preached a little among murmured choruses of ''Amen'' and ''that's right brother.''
''It wasn't really sermonic as it was truly from the heart,'' said Charles Wallace of Memphis. ''He tried to convey a sense of realness, especially when it comes to human relations.''
However, some spectators said Gore wasn't as stirring as Clinton was when he became the first president to address the denomination in 1993.
''He's no Bill Clinton, he ain't no Jesse Jackson. He needs some boning up big time,'' said one man, who declined to give his name.
Gore vowed to continue to fight for civil rights and against discrimination, but he said the battle must also come from within the communities.
''We have the capacity to transcend it (discrimination),'' Gore said, ''but not if we pretend it is not present.''
One issue of discrimination Gore said he would address if elected president was the delay in nominations of black federal appointees.
He told stories about his late father, Al Gore Sr., who was one of only three Southern U.S. senators who refused to sign the ''Southern Manifesto'' opposing desegregation.
''He's certainly from good stock,'' said Presiding Bishop Chandler Owens of the vice president.
Gore sited several passages from King's famous speech on April 3, 1968, when he promised followers that they would get to the promised land, though ''I may not get there with you.'' He was gunned down the next day on a balcony at a Memphis motel.
Among the passages Gore chose was one in which King said if he could see any period in human history it would be the latter half of this century. Gore said that was ''the time he felt God was moving in the affairs of men and women to bring about profound change in our hearts.
''That prophecy has continued to unfold and I believe it is time for us to look at what lies ahead, to what this new era will bring to us and decide what kind of future we would like to have for our children and grandchildren,'' he said.
Clinton and Gore have long enjoyed strong backing from blacks, but the vice president wants to make sure that support doesn't erode as he battles former Sen. Bill Bradley for the Democratic nomination.
Bradley has made racial unity a centerpiece of his campaign, saying it is ''the defining moral issue of our time.'' Still, Gore consistently outpolls Bradley among black voters, especially in the South.
© Copyright 2002 USA TODAY, a division of Gannett Co. Inc.


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Triple Mini Cattleya Orchid Liquid Illusion Silk Flower Arrangement
Cherish the beauty and serenity of this multi-colored orchid display. The glistening green foliage is the perfect compliment to the soft blooms (and buds) of the flowers. Displayed in an attractive glass vase, the stems are enclosed in bamboo supports immersed in artificial water. These authentic looking flowers make the perfect gift for any occasion.
95% of orders for this product ships within 2 business days plus transit time.
Product Features:


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What's funnier than someone in a bear suit? A bear in a Batman suit. Or a ninja suit. Or an Ewok suit.
Pop reader JasonC told me about Julian Santiago's website, where the artist is posting 365 bear illustrations this year. The images are too adorable for words; you'll want to bookmark this one for daily inspiration (er, insbearation?).
A few faves:
Look, it's Captain Ameribear!
This one is inspired by Brobee from Yo Gabba Gabba!:
And here's the bear Santiago calls a "B-wok":
New bears are posted daily.
Whitney has been writing about pop culture for USA TODAY since 1999, when Pop Candy began as a weekly column. Her interests include music festivals, mustaches, Lost, comic books and Andy Warhol. She lives in New York City. More about Whitney | E-mail Whitney


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2nd Annual London Surf Film Festival 11-14 October 2012
This October sees the 2nd Edition of the London Surf / Film Festival touch down. An international celebration of the very cream of surf culture, the London Surf / Film Festival brings together film, art, photography and lifestyle from waveriding's most exciting creatives.
The independent festival is a showcase international surfing's best feature films, documentaries and indie films plus the pick of short film productions from Britain and Ireland's homegrown talents. Accompanied by Q&A, talks and workshops with legends of the surf as well as a gallery show, live music sessions and more, the London Surf / Film Festival is the 'must attend' event for purveyors or the glide.
More information is available on the official site.


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Apeldoorn by Studio Makkink & Bey
Jurgen Bey was asked by the ROC professional training school in Apeldoorn
in the Netherlands to design a classroom interior. The
classroom walls, floor and furniture are decorated with images taken from
reference books used at the school. Everything in the classroom is coloured
either white or grey. Spaces are divided by moveable hanging screens that
are also printed with technical patterns. The floors are made with Senso
Freeze, a unique flooring system based on transparent resin, allows any
digital photo to be layed-out on the floor and 'frozen' into it. The system
is colour-fast and highly wear resistant.


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Pet Bone Toy
Eco-friendly durable bone toys in all the spring colors! These will look great in any pampered pets Easter Basket!
Two colorful paws up for these pet food scoops. Not only are they fun but they give you just the right amount of food for your pets hungry tummy (pne cup per paw).Tags: animals, Cats, pet lovers, Dog, scoops, dog products, pet products, Pet Owners, animal productsAdded 10 Years Ago from Libastyle
Add a little fun to your pets food! These dog and cat inspired scoopers are a great gift for any pet owner.Tags: animals, Cats, Dog, pets, scoops, spoons, dog products, pet foods, kitchen toolsAdded 10 Years Ago from Libastyle


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NEW YORK (Dec. 17)
The Israel Bond drive in New York alone has resulted this year in sales exceeding $10,300,000, topping the total 1960 figure, it was announced here today by Ira Guilden, chairman of the Greater New York Israel Bond Committee.
A capacity audience of 18,000 bond purchasers will fill Madis on Square Garden tomorrow night, for the second of the Israel Bond Chanukah Festivals this year, Mr. Guilden said. A third performance of the Chanukah Festival has been scheduled for the evening of January 1; Tickets to the event are offered free to those who purchase Israel bonds.


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Archive for Thursday, March 7, 2013
THS wrestlers earn academic honors
March 7, 2013
The Tonganoxie High wrestling team earned an honorable mention on the Kansas Wrestling Coaches Association's 2012-13 academic state championship list. To qualify for an honorable mention, the team had to have a cumulative grade point average between 2.8 and 3.49.
A pair of seniors were also honored individually. Thomas Miller (220 pounds) earned first-team honors, meaning he owns a 3.5 or higher GPA. Gus Dent (170) was an honorable mention with a GPA between 3.0 and 3.49.
Miller and Dent were both state qualifiers this year. Miller finished the season with a 38-11 record and took sixth at state, while Dent went 19-13 in his only season as a varsity wrestler.
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When it comes to food preparation, proper hygiene is of the utmost importance. Washing hands and tools may seem like simple measures, but if we didn't practice these steps of basic hygiene, diseases would spread at a much greater rate than they do currently.
We didn't always know about the importance of protecting ourselves from bacteria. In some places, people still don't take
Indiantown FL 34956 personal hygiene seriously when it comes to food. To standardize the practices involving food preparation, the government has implemented health codes and inspections ensuring that the public is safe from consuming harmful bacteria along with their food.
These regulations state that professional food handlers must be trained in food hygiene techniques and must always implement then when on the job. Many food handlers Indiantown FL 34956 are required to attend classes and achieve a certification before being allowed to prepare food.
In some cases, each employee requires certification, while in others, the restaurant as a whole must be certified. The rules regarding certification vary according to the geographical location.
If you operate a food service of some type, it's a good idea to call the local health board and inquire
Indiantown FL 34956 about the certifications that you need to operate. These calls don't provoke the health board into inspecting your place of business, so don't worry that you're making them suspicious.
It's more likely that they'll appreciate that you're trying to do the right thing, and will be more cooperative with you as time goes on.
Food hygiene certifications require that you take at least one Indiantown FL 34956 class, or more likely, a series of classes. These classes are usually available at a local college, online, or at a night school.
When trying to decide which classes to take, examine the topics that they cover to be sure that they match up with your needs. The rules regarding certifications can vary, even from one county to another in some cases. Make Indiantown FL 34956 sure you understand what's required of you so there's no confusion later on.
food handler test online…food handler test online


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What's Bill Sammon's Role At Fox/GOP Debate?
Blog ››› ››› OLIVER WILLIS
Several pictures on the Facebook page for Special Report show Fox News Washington managing editor Bill Sammon at tonight's Republican presidential primary debate in South Carolina. It's unclear what role Sammon has with Fox's debate preparations and coverage. What is clear is Sammon lacks any credibility to manage a news bureau, much less a debate.
In recent months, Media Matters has revealed that Sammon:
- Admitted that he lied on-air during coverage of the 2008 election, speculating "about whether Barack Obama really advocated socialism" while he privately believed that the allegation was "rather far-fetched."
- Described then-Senator Obama's "views on the white race" as "fairly controversial"
- Fundraised for conservative causes
- Instructed Fox staffers on how to slant coverage of Obama's Cairo speech, making note that Obama didn't use the words "terror," "terrorist" or "terrorism" -- despite the fact that Obama discussed denouncing and confronting Al Qaeda and other "violent extremists who pose a grave threat to our security"
These (and other) instances of Sammon slanting Fox's "straight news" coverage demonstrate an allegiance to political advocacy over journalistic ethics. Will Sammon strike again in South Carolina?
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- Elections
- Network/Outlet
- Fox News Channel
- Person
- Bill Sammon
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