Summary

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Published: March 18, 2017
doi:

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( 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 )允许高盐洗涤(邻近的NaCl的溶解?…

Disclosures

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 

References

  1. 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).
  2. 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).
  3. Todd, A. K., Johnston, M., Neidle, S. Highly prevalent putative quadruplex sequence motifs in human DNA. Nucleic Acids Res. 33 (9), 2901-2907 (2005).
  4. Huppert, J. L., Balasubramanian, S. Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33 (9), 2908-2916 (2005).
  5. Bedrat, A., Lacroix, L., Mergny, J. L. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 44 (4), 1746-1759 (2016).
  6. 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).
  7. Huppert, J. L., Balasubramanian, S. G-quadruplexes in promoters throughout the human genome. Nucleic Acids Res. 35 (2), 406-413 (2007).
  8. 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).
  9. Eddy, J., et al. G4 motifs correlate with promoter-proximal transcriptional pausing in human genes. Nucleic Acids Res. 39 (12), 4975-4983 (2011).
  10. 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).
  11. 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).
  12. 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).
  13. Giri, B., et al. G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA. Nucleic Acids Res. 39 (16), 7161-7178 (2011).
  14. 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).
  15. 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).
  16. 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).
  17. 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).
  18. 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).
  19. 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).
  20. Yoo, J. S., et al. DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation. PLoS Pathog. 10 (3), e1004012 (2014).
  21. 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).
  22. 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).
  23. 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).
  24. 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).
  25. 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).

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Cite This Article
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).

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