Revision as of 17:05, 17 December 2020

TteUvrD helicase

Introduction

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines.

This TteUvrD helicase is a part of helimerase protein complex, which could be used with the aim to improve HDA method.

Biology

DNA helicases are nucleic acid-dependent ATP-ases, that separate double-stranded oligonucleotides. They are divided into six different superfamilies (SF) based on their specific structural domains^[1]. Helicases also could be categorized based on its mechanic differences. In this nomenclature, these enzymes could be classified according to their translocation polarity. Helicases with 3‘ → 5‘ translocation polarity are type A, while helicases with 5‘ → 3‘ are type B. Nonetheless, helicases could also be divided by their substrate specificity. In this classification, there are two different groups – α and β, which accordingly translocates single-stranded or double-stranded molecules^[2].

Figure 1. Bifunctional protein helimerase. TteUvrD - thermostable UvrD helicase, BstPol - Bst polymerase I large fragment, L1 - linker between WZB1/WZA2 coiled-coil amino acid structures, WZA2 - coiled-coil amino acid structure, fused with TteUvrD, WZB1 - coiled-coil amino acid structure, fused with BstPol, 10xHisTag - TteUvrD purification tag, Maltose binding protein (MBP) - TteUvrD purification tag, StrepII tag - BstPol purification tag.

The most essential role for helicases is DNA replication, where double-stranded DNA is being separated into single strands by generating energy from ATP hydrolysis. After this process single stranded DNA molecule could be easily amplified by DNA polymerases^[3]. Nowadays this process could be easily mimicked by novel isothermal helicase dependent amplification method. Usually, in this assay, two complementary DNA strands are being separated with UvrD helicase (EC: 3.6.4.12). When newly generated ssDNA is being coated with single-stranded DNA-binding proteins. The next step of HDA process is the hybridization of site-specific primers to each ending border of ssDNA. After this hybridization the elongation process performed by DNA polymerase, starts^[3]. The biggest advantage of this HDA reaction is its specificity as well as the ability to perform the reaction at a constant temperature.

However, for a long time in isothermal HDA assay, which mimics the replication fork, E. coli UvrD helicase (82 kDa) was used due to its ability to unwind blunt-ended dsDNA fragments. In HDA assay, this helicase, which belongs to the superfamily 1 (SF1), requires additional proteins such as MutL and single-stranded binding protein (SSB) to keep DNA separated during HDA reaction^[3]. Thus, by using UvrD helicase, the HDA reagent composition becomes very complex. In addition to this, the protein itself is mesophylic, which lets to perform the reaction only at 37 °C. This trait increases the possibility to obtain unspecific DNA fragments, which can cause false-positive results in further analysis^[4].

According to the latest research, with the effort to increase HDA sensitivity and efficiency, thermostable uvrD homolog (TteUvrD), obtained from thermophilic bacteria Thermoanaerobacter tengcongensis was purified and characterised4. Studies have shown, that the optimal temperature for TteUvrD activity is 55°C. It was measured by analysing ATP hydrolysis during the ATPase assay. Nonetheless, by observing the stability of this enzyme, it was determined, that the optimal temperature for TteUvrD helicase activity is above 70°C^[4].

Another important characteristic of TteUvrD helicase is its unwinding activity. Based on previous research, the activity of TteUvrD was analyzed by measuring the displacement of a radio-labeled-24DNA strand. Research has shown that TteUvrD is able to displace blunt-ended and 5‘-ssDNA tailed duplex. However, researchers determined, that event if TteUvrD prefers 3‘ssDNA tailed duplexes, its unwinding activity is much lower than seen in E. coli UvrD helicase^[4].

Nonetheless, one of the biggest advantages of TteUvrD is its capability to be active at a higher temperature. Regarding to previously mentioned HDA method, the higher reaction temperature is crucial by improving this assay specificity and sensitivity. Also, during this research, scientists showed, that by using TteUvrD helicase for HDA assay, there is no need for accessory proteins such as SSB or MutL^[4].

However, even if HDA method is sensitive and specific, it is able to amplify shorter than 200 bp sequences^[3]. This problem was solved by fusing TteUvrD helicase with Bst polymerase I large fragment (BBa_K3416003) through coiled-coil interaction (Fig. 1). Such fusion increases the speed and synthesis of new strands, which makes HDA reaction more specific and efficient. Based on similar researches, scientists have shown, that coordination between TteUvrD helicase and BstPol could increase specificity and efficiency of the HDA reaction^[5].

20 Previously mentioned proteins BstPol and TteUvrD are physically linked together by using coiled-coil structure. TteUvrD helicase is fused with one part of this structure, WinZip-A2 (WZA2), through the linker L1 and is possessed in the N-terminal end of the sequence. The research has shown that this type of coiled-coils structures provides predictable tertiary structure and stability^[6].

The most important feature of these coiled-coil amino acid sequences is seven-residue repeat (abcdefg)_n, where first (a) and fourth (d) positions need to be occupied by hydrophobic amino acids by the means to determine the oligomerization state. In parallel residues at seventh (g) and the succeeding fifth (e) positions forms hydrogen-bonded ionic interactions7. In our case these coiled coils was constructed based on c-Jun/c-Fos and GCN4 Leucine zippers, where parallel dimer is formed with the leucine in d sites and valine in a sites^[5],^[7]. According to the research, the most stable heterodimer is formed with a T_M of 63 °C, where binding affinity constant reached Kd of 4.5 nM^[5].

Results

Firstly gene sequence WZA2-L1-TteUvrD was cloned into pETDuet vector. This protein was fused with a 10xHis tag and a maltose binding protein (MBP) at N terminus.

The optimal expression conditions for WZA2-L1-TteUvrD was observed in E. coli BL21(DE3) strain after 16 hours induction with 1 mM IPTG at 30 °C. Protein was purified under native conditions. WZA2-L1-TteUvrD protein was purified by using Ni-NTA affinity column. The concentration of recombinant protein observed after purification under native conditions from 1 L medium reached 5.5 mg/ml. However, as seen in SDS-PAGE gel, further optimizations procedures are needed to remove impurities (Fig. 2).

To verify that WZA2-L1-TteUvrD and WZB1-L1-BstPol proteins are stable at high temperatures we performed fluorescence thermal shift assay by utilizing a thermodynamic model9. This measurement is based on protein property to unfold upon increasing temperature of environment and solvatochromic property of fluorescence dye to fluorescent more intensively in the hydrophobic environment^[4]. The results (Fig. 3) show that TteUvrD protein is stable at high temperatures, thus letting us use it in activity assays. Also, according to obtained melting temperatures (T_M) WZA2-L1-TteUvrD protein denaturation starts at 61.1°C.

According to obtained enzymes stability, data kinetic experiments were performed. WZA2-L1-TteUvrD activity was measured based on SYBR GreenI fluorescent dye, which emits fluorescence upon binding with double-stranded DNA. Otherwise, if helicase separates dsDNA, when fluorescence declines proportionally7. As shown in Fig. 4A and Fig. 4B the bigger amount of WZA2-L1-TteUvrD protein is in the assay, the more dsDNA is unwinded.

Sequence and Features

References

1. ↑ Singleton, M. R., Dillingham, M. S. & Wigley, D. B. Structure and mechanism of helicases and nucleic acid translocases. Annual Review of Biochemistry vol. 76 23–50 (2007).
2. ↑ Huttner, D. & Hickson, I. D. Helicases. in Brenner’s Encyclopedia of Genetics: Second Edition 406–408 (Elsevier Inc., 2013). doi:10.1016/B978-0-12-374984-0.00687-2.
3. ↑ ^{3.0 3.1 3.2 3.3} Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Reports 5, 795–800 (2004).
4. ↑ ^{4.0 4.1 4.2 4.3 4.4} Curti, E., Smerdon, S. J. & Davis, E. O. Characterization of the helicase activity and substrate specificity of Mycobacterium tuberculosis UvrD. Journal of Bacteriology 189, 1542–1555 (2007).
5. ↑ ^{5.0 5.1 5.2} Muller, K.M., Arndt, K.M., Albert, T. Protein fusions to coiled-coil domains. Methods Enzymol. 328, 261-282.
6. ↑ Woolfson, D. N. Coiled-coil design: Updated and upgraded. Sub-Cellular Biochemistry 82, 35–61 (2017)
7. ↑ Matulis, D., Kranz, J. K., Salemme, F. R. & Todd, M. J. Thermodynamic Stability of Carbonic Anhydrase: Measurements of Binding Affinity and Stoichiometry Using ThermoFluor. (2005) doi:10.1021/bi048135v.