Review



wave hplc nucleic acid fragment analysis system  (Transgenomic)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 90
      Buy from Supplier

    Structured Review

    Transgenomic wave hplc nucleic acid fragment analysis system
    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    Wave Hplc Nucleic Acid Fragment Analysis System, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wave+hplc+nucleic+acid+fragment+analysis+system/wave+hplc+nucleic+acid+fragment+analysis+system/pmc05204125-416-27-41
    Average 90 stars, based on 1 article reviews
    wave hplc nucleic acid fragment analysis system - by Bioz Stars, 2026-10
    90/100 stars

    Images

    1) Product Images from "Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile"

    Article Title: Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile

    Journal: Open Biology

    doi: 10.1098/rsob.160272

    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    Figure Legend Snippet: A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.

    Techniques Used: Residue, Binding Assay, Sequencing, High Performance Liquid Chromatography, Activity Assay, Mutagenesis, Synthesized

    Related Articles

    High Performance Liquid Chromatography:

    Article Title: Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines
    Article Snippet: The Sephadex G-25 microspin columns were purchased from Amersham (Piscataway, NJ, USA). .. The WAVE HPLC Nucleic Acid Fragment Analysis System, HPLC Buffer A (0.1 M triethylammonium acetate, pH 7.0), HPLC Buffer B (0.1 M triethylammonium acetate, 25% acetonitrile v/v) and the DNA Sep HPLC column were obtained from Transgenomic (Omaha, NE, USA). .. All oligonucleotide templates used in this study were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA).

    Article Title: Interactions between helicase and primase are crucial for DNA replication in the enteropathogen Clostridium difficile
    Article Snippet: .. For the denaturing HPLC analyses, a gradient 0–8.8% v/v acetonitrile over 16 min was used to obtain optimal separation of primer products and ssDNA templates on a WAVE HPLC Nucleic Acid Fragment Analysis System equipped with a DNA Sep HPLC column (Transgenomic; Omaha, NE). ..

    Article Title: Thermally denaturing high-performance liquid chromatography analysis of primase activity.
    Article Snippet: Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics.. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting.. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized.

    Article Title: Staphylococcus aureus primase has higher initiation specificity, interacts with single-stranded DNA stronger, but is less stimulated by its helicase than Escherichia coli primase.
    Article Snippet: NTPs were purchased from Promega (Madison, WI). dNTPs and ddNTPs were obtained from Roche Molecular Biosystems (Mannheim, Germany). .. Primer lengths and quantities were measured on a WAVE HPLC Nucleic Acid Fragment Analysis System with a DNASep HPLC column from Transgenomic (Omaha, NE). ..

    Article Title: Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile
    Article Snippet: .. For the denaturing HPLC analyses, a gradient 0–8.8% v/v acetonitrile over 16 min was used to obtain optimal separation of primer products and ssDNA templates on a WAVE HPLC nucleic acid fragment analysis system equipped with a DNA Sep HPLC column (Transgenomic; Omaha, NE, USA). ..

    Article Title: Allosteric regulation of the primase (DnaG) activity by the clamp-loader (tau) in vitro.
    Article Snippet: The Sephadex G-25 microspin columns were purchased from Amersham (Piscataway, NJ). .. Primer lengths and quantities were measured using a WAVE HPLC Nucleic Acid Fragment Analysis System with a DNA Sep HPLC column, buffer A (0.1 M triethylammonium acetate, pH 7.0) and buffer B (0.1 M triethylammonium acetate, 25% acetonitrile v/v) that were obtained from Transgenomic (Omaha, NE). .. The 23-mer oligonucleotide used in this study consisted of the sequence 5′-CAGA(CA)5XYZ (CA)3-3′ where XYZ was CTA, the preferred trinucleotide for G. stearothermophilus DnaG (Thirlway and Soultanas, 2006).

    Article Title: Myricetin inhibits Escherichia coli DnaB helicase but not primase.
    Article Snippet: .. After incubation at 30 C for 1 h, the samples were desalted through a Microspin G-25 column (Amersham, Piscataway, NJ) and then separated by thermally denaturing HPLC on a WAVE HPLC Nucleic Acid Fragment Analysis System with a DNASep HPLC column from Transgenomic (Omaha, NE). .. The data were fit to the indicated equations using Prism 4 for Macintosh (GraphPad Software, San Diego, CA).

    Incubation:

    Article Title: Myricetin inhibits Escherichia coli DnaB helicase but not primase.
    Article Snippet: .. After incubation at 30 C for 1 h, the samples were desalted through a Microspin G-25 column (Amersham, Piscataway, NJ) and then separated by thermally denaturing HPLC on a WAVE HPLC Nucleic Acid Fragment Analysis System with a DNASep HPLC column from Transgenomic (Omaha, NE). .. The data were fit to the indicated equations using Prism 4 for Macintosh (GraphPad Software, San Diego, CA).



    Similar Products

    wave hplc nucleic acid fragment analysis system  (Transgenomic)
    90
    Transgenomic wave hplc nucleic acid fragment analysis system
    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    Wave Hplc Nucleic Acid Fragment Analysis System, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wave+hplc+nucleic+acid+fragment+analysis+system/wave+hplc+nucleic+acid+fragment+analysis+system/pmc05204125-416-27-41
    Average 90 stars, based on 1 article reviews
    wave hplc nucleic acid fragment analysis system - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    denaturing hplc wave nucleic acid fragment analysis system  (Transgenomic)
    90
    Transgenomic denaturing hplc wave nucleic acid fragment analysis system
    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    Denaturing Hplc Wave Nucleic Acid Fragment Analysis System, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wave+hplc+nucleic+acid+fragment+analysis+system/wave+hplc+nucleic+acid+fragment+analysis+system/pm24150844-31-12-20
    Average 90 stars, based on 1 article reviews
    denaturing hplc wave nucleic acid fragment analysis system - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    reversed-phase hplc column wave nucleic acid fragment analysis system  (Transgenomic)
    90
    Transgenomic reversed-phase hplc column wave nucleic acid fragment analysis system
    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    Reversed Phase Hplc Column Wave Nucleic Acid Fragment Analysis System, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wave+hplc+nucleic+acid+fragment+analysis+system/wave+hplc+nucleic+acid+fragment+analysis+system/pm19460837-53-10-19
    Average 90 stars, based on 1 article reviews
    reversed-phase hplc column wave nucleic acid fragment analysis system - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    ip-rp-hplc system transgenomic wave nucleic acid fragment analysis system  (Transgenomic)
    90
    Transgenomic ip-rp-hplc system transgenomic wave nucleic acid fragment analysis system
    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    Ip Rp Hplc System Transgenomic Wave Nucleic Acid Fragment Analysis System, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wave+hplc+nucleic+acid+fragment+analysis+system/wave+hplc+nucleic+acid+fragment+analysis+system/pm17385798-34-1-6
    Average 90 stars, based on 1 article reviews
    ip-rp-hplc system transgenomic wave nucleic acid fragment analysis system - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    d-hplc wave nucleic acid fragment analysis system 3500  (Transgenomic)
    90
    Transgenomic d-hplc wave nucleic acid fragment analysis system 3500
    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.
    D Hplc Wave Nucleic Acid Fragment Analysis System 3500, supplied by Transgenomic, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wave+hplc+nucleic+acid+fragment+analysis+system/3500ht+wave+dna+fragment+analysis+system/pm17096675-45-16-24
    Average 90 stars, based on 1 article reviews
    d-hplc wave nucleic acid fragment analysis system 3500 - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier

    Image Search Results


    A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.

    Journal: Open Biology

    Article Title: Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile

    doi: 10.1098/rsob.160272

    Figure Lengend Snippet: A lysine residue affect trinucleotide specificity of CD1454. ( a ) Alignment of the α2–α3 region of the zinc-binding domain of primases with characterized trinucleotide specificity. Predicted secondary structure (α helix in grey, β sheet in white) is indicated above the alignment. Ftula, Francisella tularensis; Ecoli, Escherichia coli ; Ypest, Yersinia pestis ; Paeru, Pseudomonas aeruginosa ; Cdiff, Clostridium difficile ; Aaeol, Aquifex aeolicus ; Saure, Staphylococcus aureus ; Cperf, Clostridium perfringens ; Gstea, Geobacillus stearothermophilus ; Banth, Bacillus anthracis ; Bsubt, Bacillus subtilis . Highlighted are the zinc coordinating residues of the CHC2 zinc-binding motif (yellow), and the adjacent histidine (green) or lysine (red) residues. Sequence conservation is indicated below the alignment. ( b ) Ribbon representation of the CD1454 primase zinc-binding domain indicating the zinc coordinating residues (yellow) and the location of the lysine residue (red). ( c ) Thermally denaturing high-performance liquid chromatography analysis of primase activity of wild-type (WT) and K70H mutant CD1454 primase protein with the CCC and CTA containing templates. The primase (WT or K70H mutant) and initiation trinucleotide in the single-stranded 23-mer template used is shown in the upper left corner of the respective chromatogram. The numbers in the panel to the right of the chromatograms denote the total peak area for the RNA primer products synthesized and shown in the associated chromatogram to the left. The mutation affects RNA primer synthesis from both preferred 5′-d(CCC) and non-preferred 5′-d(CTA) trinucleotides but shows the strongest increase in activity from the 5′-d(CTA) trinucleotide.

    Article Snippet: For the denaturing HPLC analyses, a gradient 0–8.8% v/v acetonitrile over 16 min was used to obtain optimal separation of primer products and ssDNA templates on a WAVE HPLC nucleic acid fragment analysis system equipped with a DNA Sep HPLC column (Transgenomic; Omaha, NE, USA).

    Techniques: Residue, Binding Assay, Sequencing, High Performance Liquid Chromatography, Activity Assay, Mutagenesis, Synthesized