Method Article
G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells VSports. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.
Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat VSports app下载. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.
G4 structures are highly stable nucleic acid secondary structures that form within guanine-rich regions of DNA and RNA. G4 structures are stabilized via Hoogsteen-bonding interactions and coordinate bonding within the central cavity with monovalent cations (i. e V体育官网. K+ and Na+) that significantly contribute to the remarkable thermal stability of G4 structures1,2. Early bioinformatics studies suggested that the human genome contains >375,000 “potential G4-forming motifs”3,4. More recent study esti.
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1 V体育安卓版. Preparation of G4-DNA Structures to be Used for the Purification of rG4R1 (Formation of Biotinylated G4-DNA G-Quadruplex).
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This protocol (Figure 1) routinely yields nearly pure, catalytically active rG4R1. As a measure of enzymatic activity, it is typically observed that 50% of 0. 2 pmol of TAMRA-labeled tetramolecular G4-DNA is converted into monomers within the 0. 2 - 0 VSports最新版本. 013 µL range of rG4R1, as assayed by the G4 activity assay outlined above (Figure 2). Coomassie staining of purified rG4R1 indicates a single band at the expected 120 kDa size, with a minor contaminating band at ~75 kDa, which may repres.
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This protocol represents a highly efficient expression, purification, and quantification scheme for the isolation of the DHX36 gene product, G4-Resolvase1 (G4R1, also called RHAU and DHX36) (Figure 1). This protocol utilizes two purification steps: His-tag affinity purification on cobalt affinity beads and enzymatic purification on G4-DNA-conjugated beads. The latter step is unique in that it takes advantage of the tight affinity, high specificity, and catalytic unwinding activity that rG4R1 has for G4 s.......
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The authors have nothing to disclose.
We would like to thank our funding sources, including a generous gift from the Ware Foundation (to J.P.V.), The National Institutes of HealthGrant T32-CA079448 (to P.J.S.), and Ball State University startup funds (to P. J. S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| TriEx4-DHX36 plasmid | Addgene | 68368 | |
| Rosetta2(DE3)plysS competent cells | Novagen | 71403-4 | |
| S.O.C medium | Thermo Fisher Scientific | 15544034 | |
| Difco Terrific Broth | Becton Dickinson | 243820 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Chloramphenicol | Sigma-Aldrich | C1919 | 35 µg/mL in bacterial plates/large cultures |
| Carbenicillin (plant cell culture tested) | Sigma-Aldrich | C3416 | 50 µg/mL in bacterial plates/large cultures |
| Isopropyl β-D-1-thiogalactopyranoside (IPTG) | Sigma-Aldrich | I6758 | |
| Lysozyme (from chicken egg white) | Sigma-Aldrich | L6876 | |
| 1 M Tris-HCl, pH 8 | Universal Scientific Supply Co. | 1963-B | or from standard source |
| 1 M Tris-HCl, pH 7 | Universal Scientific Supply Co. | 1966 | or from standard source |
| 1.5 M Tris-HCl, pH 8.8 | For casting resolving gel (for protein quantitation gel); From standard source | ||
| 1 M Tris-HCl, pH 6.8 | For casting stacking gel (for protein quantitation gel); From standard source | ||
| 1 M Tris-Acetate, pH 7.8 | Universal Scientific Supply Co. | 1981 | or from standard source |
| 70% Ethanol | From standard source | ||
| Magnesium chloride (1 M solution) | Life Technologies | AM9530G | |
| Sodium chloride | Sigma-Aldrich | S7653 | |
| Sodium acetate | Sigma-Aldrich | S8750 | |
| 20x SSC | Universal Scientific Supply Co. | 1665 | or from standard source |
| β-mercaptoethanol (2-BME) | Sigma-Aldrich | 63689 | |
| Protease inhibitor cocktail | Sigma-Aldrich | P8849 | |
| Leupeptin hemisulfate | Sigma-Aldrich | L8511 | |
| Streptavidin paramagnetic beads | Promega | Z5482 | |
| 0.5 M EDTA, pH 8 | Universal Scientific Supply Co. | 0718 | or from standard source |
| 0.2 M EDTA, pH 6 | Universal Scientific Supply Co. | From standard source; initially adjust pH with NaOH, then adjust pH back down with HCl. | |
| A-lactalbumin (Type 1 from bovine milk) | Sigma-Aldrich | L5385 | |
| Cobalt metal affinity beads | Clonetech | 635502 | |
| L-Histidine | Sigma-Aldrich | H8000 | |
| Acetic acid, glacial | Fisher Scientific | A38-500 | |
| Adenosine 5'-Triphosphate (from bacterial source) | Sigma | A7699 | |
| 40% acrylamide/Bis solution (37.5:1) | Biorad | 161-0148 | |
| Glycine | Sigma-Aldrich | 50046 | to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS) |
| 10% Sodium dodecyl sulfate (SDS) | Universal Scientific Supply Co. | 1667 | to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS); or from standard source |
| 10x TBE | Sigma-Aldrich | 11666703001 | or from standard source |
| Tris base | Fisher Scientific | BP152-1 | to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS); From standard source |
| TEMED | Sigma-Aldrich | 411019 | |
| Ammonium persulfate (APS) | Sigma-Aldrich | A3678 | |
| Broad Range Protein MW markers | Promega | V8491 | |
| Biotinylated Z33 oligo ("Z33-Bio") | Oligos Etc | 5’ AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT-biotin 3’ | |
| TAMRA-Z33 oligo ("Z33-TAM") | Oligos Etc | 5’ TAMRA-AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT 3’ | |
| Fluor-coated TLC plate | Life Technologies | AM10110 | |
| Ficoll | Sigma-Aldrich | F2637 | 30% in H2O |
| Coomassie R-250 | Sigma-Aldrich | 27816 | |
| Methanol | Fisher Scientific | A412 | |
| Multiband UV lamp | Capable of emitting UV light at 365 nm | ||
| Table-top centrifuge (with swinging bucket rotor) | Capable of being cooled to 4 °C | ||
| Microcentrifuge | Capable of being cooled to 4 °C | ||
| Digital Sonfier | Branson | Or equivalent capable of delivering sonication pulses (30% amplitude, 2 s ON/2 s OFF) | |
| 50 °C water bath | For formation of Z33 into quadruplex | ||
| 37 °C incubator for bacteria | For bacterial transformations and initial overnight growth of large cultures of Rosetta2 E. coli transformed with TriEx4-DHX36 | ||
| 37 °C/14 °C shaking incubator for bacteria | For growth and protein induction of large cultures of Rosetta2 E. coli transformed with TriEx4-DHX36 | ||
| Spectrophotometer | capable of reading OD600; capable of reading oligomer concentrations based on base sequence (such as Biorad SmartSpec 3000) | ||
| Thermometer | From standard source | ||
| PCR strip tubes | From standard source | ||
| 15 and 50 mL centrifuge tubes (polypropylene) | From standard source | ||
| Microcentrifuge tubes (2.0 mL) | From standard source | ||
| 500 mL centrifuge bottles (polypropylene) | Thermo Scientific | 3141-0500 | |
| Standard array of pipet tips and serological pipettes | From standard source | ||
| Gel-loading tips | From standard source | ||
| Automatic repeating pipette | For quick aliquoting of rG4R1; From standard source | ||
| Thermal cycler | From standard source | ||
| Liquid Nitrogen | From standard source | ||
| Dry ice | From standard source | ||
| Laemlli sample buffer | Biorad | 161-0737 | |
| Apparatus for running large slab gels | Biorad | We have used the Protean II xi cell apparatus from Biorad | |
| Magnet | Life Technologies | 12301D | We use a magnet from One Lambda (Now a Thermo Fisher Scientific brand); and Life is also a subsidiary of Thermo, and thus the magnet listed here should be a suitable replacement |
| Razor blades | From standard source | ||
| Filter paper and funnel | From standard source | ||
| Glass casserole dish | From standard source | ||
| Orbital shaker | From standard source | ||
| Kimwipes | From standard source | ||
| Clear sheet protectors | From standard source | ||
| Scanner and associated TWAIN software | From standard source | ||
| Image analysis software | Such as Fuji Multiguage, or equivalent | ||
| Microsoft Excel | Or equivalent |
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