滞后链DNA合成的动力学研究<em>体外</em>由T7噬菌体复制蛋白
Summary
We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.
Abstract
Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.
Introduction
DNA的复制是所有细胞生命的一个保守的特征。而复制组件的特性和数量有很大的不同，一般机制跨越进化1共享。这里，我们描述了基于凝胶的，不连续的动力学实验，使用的放射性底物，旨在通过T7启动复制体的组分理解体外滞后链DNA合成的动力学。 T7噬菌体的复制机制是很简单的，仅由四种蛋白质（DNA聚合酶，GP5，其持续合成因子， 大肠杆菌硫氧还蛋白，双官能引发酶-解旋GP4，和单链DNA结合蛋白，GP2的。 5）2。这一特性使得它来研究参与DNA复制保守的生化机制有吸引力的模型系统。
特别强调的是由T7 DNA形成的核糖核苷酸引物引发酶，一个critica升步骤中的DNA合成的起始。此外，还可以通过将其包括在反应混合物中检查这些引物由T7 DNA聚合酶或其他复制蛋白的用法。 T7启动引发酶 – 解旋酶，GP4，催化的引物DNA的合成在称为引发酶识别位点，或者PRS特定的DNA序列的形成，（5'-GTC-3'）。在PRS胞嘧啶是隐蔽，它是用于识别站点的必需的，但它不被复制到产品3。在引物合成的第一步由GP4涉及二核苷酸pppAC，然后扩展到三聚体，并最终官能四核苷酸引物pppACCC，pppACCA或pppACAC取决于序列在模板4的形成。这些引物然后可以通过T7 DNA聚合酶被用于启动DNA合成，这也是一个GP4辅助过程5,6。在这方面，引发酶域稳定极短的tetraribonucleotides与模板，防止其分解，以有利于保护底漆/模板到聚合酶活性位点7的方式从事DNA聚合酶。这些步骤（RNA引物的合成，引物越区切换到聚合酶和扩展）中重复多次循环复制后随链，必须与前导链的复制进行协调。
此处所描述的测定法是高度敏感的，并可以在一个温和的时间框架来执行。然而，它们是相对低的通量和必须十分小心以放射性物质的使用和处置行使。取决于在该反应进行时，人们可能会采用一种快速淬火仪在时间尺度适合用于在任一稳定或预稳态反应时间课程有意义的分析获得的样品的速度。最近，我们这里使用的描述测定提供我国证据的引物释放的从GP4的冈崎片段的开始时的引发酶域的重要性即此外，我们发现了为T7启动单链DNA结合蛋白，gp2.5调节作用的证据，在促进高效引物形成和利用8。
Protocol
Representative Results
Discussion
在执行这些实验的最重要的因素是高活性的纯化的酶的可用性。在我们与GP4的工作，例如，我们发现，在缓冲液中纯化的酶的含50％甘油的存储（20mM磷酸钾，pH 7.4，0.1毫摩尔EDTA，1毫摩尔DTT）在-20℃导致减少制备过几个月的比活性。因此，我们现在在25mM的Tris-HCl，pH值7.5，50 mM氯化钠，0.1毫摩尔EDTA，1毫TCEP纯化GP4，和10％甘油的闪冻小等份用液氮并将其存储在-80℃。在使用快速淬火流仪，特别是采?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).
Materials
Tris pre-set crystals pH 7.5 |
SIGMA | T4128 | |
| Tris base | SIGMA | 93362 | |
| L-Glutamic acid potassium salt monohydrate | SIGMA | G1501 | |
| Magnesium chloride hexahydrate | Mallinckrodt Chemicals | 5958-04 | |
| 1,4-Dithiothreitol | J.T. Baker | F780-02 | |
| Sodium Dodecyl Sulfate | MP Biomedicals | 811030 | |
| (Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate | Mallinckrodt Chemicals | 4931-04 | |
| [α-32P] CTP | PerkinElmer | BLU508H | radioactive, take protective measures |
| [γ-32P] ATP | PerkinElmer | BLU502A | radioactive, take protective measures |
| 100 mM dATP, dCTP, dGTP, dTTP | Affymetrix | 77100 | four dNTPs part of set |
| 100 mM ATP and CTP | Epicentre | RN02825 | part of NTP set |
| ssDNA constructs | Integrated DNA Technologies | N/A | custom sequences |
| methanol | Macron Fine Chemicals | 3017-08 | |
| formamide | Thermo Scientific | 17899 | |
| bromophenol blue | SIGMA | B8026 | |
| cylene xyanol | SIGMA | X4126 | |
| acrylamide | SIGMA | A9099 | Toxic |
| N,N′-Methylenebisacrylamide | SIGMA | M7279 | Toxic |
| Urea | Boston Bioproducts | P-940 | |
| Boric acid | Mallinckrodt Chemicals | 2549 | |
| Rapid Quench-Flow Instrument | Kintek Corp. | RQF-3 | |
| Kintek Explorer | Kintek Corp. | N/A | |
| Kaleidagraph | Synergy Software | N/A |
Riferimenti
- Kornberg, A., Baker, T. A. . DNA replication. , (1992).
- Hamdan, S. M., Richardson, C. C. Motors, switches, and contacts in the replisome. Annu Rev Biochem. 78, 205-243 (2009).
- Mendelman, L. V., Notarnicola, S. M., Richardson, C. C. Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo. Proc Natl Acad Sci U S A. 89 (22), 10638-10642 (1992).
- Qimron, U., Lee, S. J., Hamdan, S. M., Richardson, C. C. Primer initiation and extension by T7 DNA primase. EMBO J. 25 (10), 2199-2208 (2006).
- Kato, M., Ito, T., Wagner, G., Richardson, C. C., Ellenberger, T. Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis. Mol Cell. 11 (5), 1349-1360 (2003).
- Kusakabe, T., Richardson, C. C. Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites. J Biol Chem. 272 (19), 12446-12453 (1997).
- Lee, S. J., Zhu, B., Hamdan, S. M., Richardson, C. C. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7. Nucleic Acids Res. 38 (13), 4372-4383 (2010).
- Hernandez, A. J., Lee, S. J., Richardson, C. C. Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome. Proc Natl Acad Sci U S A. 113 (21), 5916-5921 (2016).
- Tabor, S., Richardson, C. C. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A. 92 (14), 6339-6343 (1995).
- Kim, Y. T., Tabor, S., Bortner, C., Griffith, J. D., Richardson, C. C. Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein. J Biol Chem. 267 (21), 15022-15031 (1992).
- Frick, D. N., Richardson, C. C. DNA primases. Annu Rev Biochem. 70, 39-80 (2001).
- Lee, S. J., Richardson, C. C. Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7. J Biol Chem. 276 (52), 49419-49426 (2001).
- Cao, W., De La Cruz, E. M. Quantitative full time course analysis of nonlinear enzyme cycling kinetics. Sci Rep. 3, 2658 (2013).
- Wang, M. H., Zhao, K. Y. A simple method for determining kinetic constants of complexing inactivation at identical enzyme and inhibitor concentrations. FEBS Lett. 412 (3), 425-428 (1997).
- Johnson, K. A. Fitting enzyme kinetic data with KinTek Global Kinetic Explorer. Methods Enzymol. 467, 601-626 (2009).
- Johnson, K. A. A century of enzyme kinetic analysis, 1913. FEBS Lett. 587 (17), 2753-2766 (2013).
- Biswas, T., Resto-Roldan, E., Sawyer, S. K., Artsimovitch, I., Tsodikov, O. V. A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG. Nucleic Acids Res. 41 (4), e56 (2013).
- Sanyal, G., Doig, P. Bacterial DNA replication enzymes as targets for antibacterial drug discovery. Expert Opin Drug Discov. 7 (4), 327-339 (2012).
