对酶活性G4 Resolvase1净化用G-四DNA亲和方法
Summary
G4 Resolvase1结合G-四（G4）的结构与一个G4结合蛋白的紧密报道的亲和力，并表示在HeLa细胞中的大多数G4-DNA解旋活动。我们描述了线束的亲和力和G4-Resolvase1的ATP依赖的平仓活动，特别是净化催化活性的重组G4R1一个新的协议。
Abstract
高阶核酸结构称为G-四链（台G4，G4结构）可以在DNA和RNA的富含鸟嘌呤区形成并且是高度热稳定的。有在人类基因组> 375000推定G4形成的序列，它们在启动子区域，非翻译区（UTR）富集和端粒重复内。由于对这些结构以影响细胞过程，如复制和转录的潜力，所述细胞已演变的酶来管理它们。一种这样的酶是G4解离1（G4R1），将其生化共同特征在于我们的实验室和长峰等。并发现极紧到两个G4-DNA和G4-RNA（K d。在低PM范围）结合。 G4R1是多数在HeLa细胞裂解物的G4分辨活性的来源，并一直牵连发挥端粒代谢，淋巴开发，基因转录，造血和免疫监视作用。 EF英孚的能力ficiently表达和纯化催化活性G4R1是重要的，针对有意获得进一步的深入了解G4结构和G4-解决酶的动力学相互作用实验室。在这里，我们描述了重组G4R1（rG4R1）的提纯的详细方法。所描述的过程结合的C-末端组氨酸标记的酶的传统的基于亲和纯化在具有rG4R1的能力利用人密码子优化的细菌中表达结合和放松G4-DNA的纯化高活性酶在ATP的依赖洗脱步骤。该协议还包括其中rG4R1的酶活性是通过检查纯化的酶的放松G4-DNA的能力测得的质量控制步骤。的方法还描述了允许纯化rG4R1的量化。该协议的另类改编进行了讨论。
Introduction
G4结构是DNA和RNA的富鸟嘌呤区域内形成高度稳定的核酸的二级结构。 G4结构通过Hoogsteen碱基接合相互作用来稳定，并与一价阳离子（ 即 K +和Na +），该显著有助于G4结构1,2的显着热稳定性的中心空腔内坐标粘接。早期的生物信息学研究表明，人类基因组包含> 375000“潜在G4形成的图案”3,4。最近的研究估计表明，G4基序的数目是由2-5 5倍以上，而另一项研究预测在人类基因组6 716310不同电位G4形成的序列。 G4形成序列进化上保守的，而不是随机地分散在基因组中。 G4图案富集基因编码区，以及所有基因启动子的40％以上含有G4图案7。有趣的是，在一个基因的G4基序的富集的程度已被证明表明该基因的功能。例如，参与发展原癌基因和基因比抑癌基因8,9 G4结构显著更大的富集。
具有高的热稳定性，在整个基因组中的几乎无处不在，并且显著影响主要的细胞过程的潜力，这是令人吃惊地发现，细胞已经发展酶来管理这些结构。一种这样的酶是G4 Resolvase1（G4R1;也称作RHAU和DHX36），我们表征为在人类（HeLa细胞）细胞10多数tetramolecular G4-DNA解析活性的来源。自那时以来，它已被证明塔吨G4R1紧密结合并催化开卷tetramolecular和单分子G4-DNA和G4-RNA与用于G4结合蛋白11，12，13中的紧密的报告的KDS。此外，G4R1的G4分辨活性已经牵涉在广泛的生物化学和细胞过程，包括端粒/端粒酶生物学11，14，15，16，转录和剪接17，18，19，20，发展21，造血21，及免疫调节22,23。用G4序列占优势的特别位于整个基因组和不同的对细胞processes该G4R1最近已经牵连用，表达和有效地净化活性高rG4R1将是极为重要的为阐明该蛋白的生化机制和行为的能力有关。
在这里，我们证明了一种新的表达而这需要rG4R1的ATP依赖性，G4-解决活动的优势，以有效地分离活性酶的纯化方案（ 图1）。该方案可以适用于纯化其它ATP依赖性核酸酶的量，酶反应的产物是不再结合的基板，如为G4R1的情况。
Protocol
1. G4-DNA结构的制备用于rG4R1（生物素G4-DNA G-四的形成）的净化订购以下的DNA寡聚体，称为Z33-生物，在1微摩尔规模：5'AAA GTG GTG ATG GTG GGG GAA GGA TTC GGA CCT生物素3'。确保该生物素部分是在该低聚物的3'末端。 准备10X G4缓冲液：450毫米的Tris-HCl pH值为8，25毫摩尔EDTA，2500毫米氯化钠。 悬浮在水250微升的Z33寡聚物（以使低聚物浓度为〜2.5毫摩尔）。加入10X G4缓冲的25μL混匀。?…
Representative Results
该协议（ 图1）经常产生几乎纯，催化活性rG4R1。作为酶活性的量度，它通常被观察到，0.2皮摩尔TAMRA标记tetramolecular G4-DNA的50％在0.2内转化成单体- rG4R1 0.013微升范围，由上述（ 图中概述的G4活性测定为测定2）。纯化rG4R1的考马斯染色在约75 kDa的，这可能代表截短rG4R1已保持其结合G4-DNA的珠和在ATP依赖洗脱能力表明在预期120 kDa的大小的单一条带，具有轻微的污?…
Discussion
该协议代表一高效表达，纯化，并定量为DHX36基因产物，G4-Resolvase1（G4R1，也称为RHAU和DHX36）（ 图1）的隔离方案。这个协议利用两个纯化步骤：关于钴亲和珠His标签的亲和纯化和酶纯化上G4-DNA缀合珠。后者步骤是在，它需要在严密的亲和力，特异性高，以及催化退绕的活动，rG4R1具有G4结构优点是唯一的。对G4结构紧亲和性（在低PM范围ķ 四 ）允许高盐洗涤（邻近的NaCl的溶解?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢我们的资金来源，包括来自洁具基金会（以JPV）一份厚礼，HealthGrant T32-CA079448（到PJS）和鲍尔州立大学的启动资金（以PJS）全国学院。该资助者在研究设计，数据收集和分析，决定发表或准备手稿没有作用。
Materials
| 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 | 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 | 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, 2s ON 2s 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 |
Riferimenti
- Qin, Y., Hurley, L. H. Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie. 90 (8), 1149-1171 (2008).
- Stegle, O., Payet, L., Mergny, J. L., MacKay, D. J., Leon, J. H. Predicting and understanding the stability of G-quadruplexes. Bioinformatics. 25 (12), i374-i382 (2009).
- Todd, A. K., Johnston, M., Neidle, S. Highly prevalent putative quadruplex sequence motifs in human DNA. Nucleic Acids Res. 33 (9), 2901-2907 (2005).
- Huppert, J. L., Balasubramanian, S. Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33 (9), 2908-2916 (2005).
- Bedrat, A., Lacroix, L., Mergny, J. L. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 44 (4), 1746-1759 (2016).
- Mendoza, O., Bourdoncle, A., Boule, J. B., Brosh, R. M., Mergny, J. L. G-quadruplexes and helicases. Nucleic Acids Res. 44 (5), 1989-2006 (2016).
- Huppert, J. L., Balasubramanian, S. G-quadruplexes in promoters throughout the human genome. Nucleic Acids Res. 35 (2), 406-413 (2007).
- Eddy, J., Maizels, N. Gene function correlates with potential for G4 DNA formation in the human genome. Nucleic Acids Res. 34 (14), 3887-3896 (2006).
- Eddy, J., et al. G4 motifs correlate with promoter-proximal transcriptional pausing in human genes. Nucleic Acids Res. 39 (12), 4975-4983 (2011).
- Vaughn, J. P., et al. The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates. J Biol Chem. 280 (46), 38117-38120 (2005).
- Booy, E. P., et al. The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary. Nucleic Acids Res. 40 (9), 4110-4124 (2012).
- Creacy, S. D., et al. G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates. J Biol Chem. 283 (50), 34626-34634 (2008).
- Giri, B., et al. G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA. Nucleic Acids Res. 39 (16), 7161-7178 (2011).
- Lattmann, S., Stadler, M. B., Vaughn, J. P., Akman, S. A., Nagamine, Y. The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme. Nucleic Acids Res. 39 (21), 9390-9404 (2011).
- Sexton, A. N., Collins, K. The 5′ guanosine tracts of human telomerase RNA are recognized by the G-quadruplex binding domain of the RNA helicase DHX36 and function to increase RNA accumulation. Mol Cell Biol. 31 (4), 736-743 (2011).
- Smaldino, P. J., et al. Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3′-Tail Sequences Are Sufficient for Tight and Complete Binding. PLoS One. 10 (7), e0132668 (2015).
- Booy, E. P., et al. The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1. Nucleic Acids Res. 42 (5), 3346-3361 (2014).
- Huang, W., et al. Yin Yang 1 contains G-quadruplex structures in its promoter and 5′-UTR and its expression is modulated by G4 resolvase 1. Nucleic Acids Res. 40 (3), 1033-1049 (2012).
- Tran, H., Schilling, M., Wirbelauer, C., Hess, D., Nagamine, Y. Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU. Mol Cell. 13 (1), 101-111 (2004).
- Yoo, J. S., et al. DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation. PLoS Pathog. 10 (3), e1004012 (2014).
- Lai, J. C., et al. The DEAH-box helicase RHAU is an essential gene and critical for mouse hematopoiesis. Blood. 119 (18), 4291-4300 (2012).
- Zhang, Z., et al. DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells. Immunity. 34 (6), 866-878 (2011).
- Kim, T., et al. Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells. Proc Natl Acad Sci U S A. 107 (34), 15181-15186 (2010).
- Booy, E. P., McRae, E. K., McKenna, S. A. Biochemical characterization of G4 quadruplex telomerase RNA unwinding by the RNA helicase RHAU. Methods Mol Biol. 1259, 125-135 (2015).
- Lattmann, S., Giri, B., Vaughn, J. P., Akman, S. A., Nagamine, Y. Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU. Nucleic Acids Res. 38 (18), 6219-6233 (2010).
Citazione di questo articolo
Routh, E. D., Creacy, S. D., Beerbower, P. E., Akman, S. A., Vaughn, J. P., Smaldino, P. J. A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1. J. Vis. Exp. (121), e55496, doi:10.3791/55496 (2017).