Effect of DNA-Binding Proteins on Terminal Deoxynucleotidyl Transferase Activity in Systems with Homopolymer Substrates

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Аннотация

In the current work we tested single strand binding protein from E. coli (EcSSB) and DNA-binding protein from S. solfataricus (Sso7d) to evaluate its effects on TdT activity for homopolymer substrates (Tn), that unable to form double helix structures. We showed a significant increase in TdT activity after adding of EcSSB even on the example of homopolymer substrates. Effects demonstrated open application of DNA binding proteins in TdT engineering and DNA-printing. The addition of EcSSB to the reaction mixture led to a significant increase in TdT activity and a shift of the reaction products towards longer oligonucleotides. The maximum effect was observed in a close to equimolar stoichiometric ratio (EcSSB)4:TdT in the presence of Mn2+ cations. In addition, the presence of Sso7d in the reaction mixture led to a slight (up to 15%) decrease in TdT activity for substrates T5 and T15 and a more pronounced decrease for T35 (up to 30%). At the same time, Co2+ cations reduced the inhibitory effect of Sso7d.The patterns and relationships established through our research have potential applications in various fields. Specifically, they can be utilized in protein engineering for the development of fusion proteins that are based on TdT. Furthermore, these findings can contribute to the advancement of novel enzymatic principles for de novo DNA synthesis.

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Авторлар туралы

A. Sachanko

Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus

Email: yantsevich@iboch.by
Белоруссия, Minsk, 220141

V. Shchur

Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus

Email: yantsevich@iboch.by
Белоруссия, Minsk, 220141

S. Usanov

Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus

Email: yantsevich@iboch.by
Белоруссия, Minsk, 220141

A. Yantsevich

Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus

Хат алмасуға жауапты Автор.
Email: yantsevich@iboch.by
Белоруссия, Minsk, 220141

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2. Fig. 1. Results of PAAG electrophoresis under denaturing conditions of samples of isolated recombinant proteins TdT (track 1), Sso7d (track 2) and EcSSB (track 3); M is the standard of molecular weights of proteins 10-250 kDa.

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3. 2. Results of electrophoresis of ring plasmid DNA (5,994 bp) in 1% agarose gel without Sso7d (track 1), in the presence of Sso7d (track 2). M is the standard of linear DNA (a); the number of homopolymer 15-dimensional oligonucleotides of various nature (nmol) bound to EcSSB (10 nmol) immobilized on Ni2+-NTA-agarose (b).

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4. 3. Dependence of TdT activity on the length of the oligonucleotide substrate in a series of Tn homopolymers.

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5. 4. Chromatograms of substrate elongation products of various lengths (T5–T50) with the participation of TdT (conc. TTR 200 microns, reaction time 30 min): Exposure time corresponding to the substrates: T5 – 5.6 min; T15 – 12.4 min; T25 – 15.2 min; T30 – 16.5 min; T45 – 18.1 min; T50 – 22.2 min.

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6. 5. The effect of Me2+ cations on the enzymatic activity of TdT.

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7. 6. Dependence of the total area of chromatographic peaks of the products (S) on the number of TdT nucleotides attached to the T5 and T15 substrates in the reaction mixture containing: 40 nM TdT, 0.2 mM TTP, 5 mM AcOK, 2 mM AsOTris (pH 7.9), 1 mM AcOMg, 0.25 mM Me2+ (indicated on according to the legend for T15), 0.4 microns of oligonucleotide (a) and a reaction mixture additionally containing 2 microns of EcSSB (b).

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8. 7. Chromatograms of reaction products at different stoichiometric ratios of EcSSB/TdT (substrate T5, tR 5.6 min, a) and EcSSB/TdT (substrate T15, tR 12.4 min, b); control: 0.2 mM TTP, 5 mM AcOK, 2 mM AsOTris (pH 7.9), 1 mM AcOMg and 0.4 microns of T5 oligonucleotide.

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9. 8. The effect of Ecsb on TdT activity for substrates of various lengths (Ecsb/TdT 5:1).

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10. 9. Effect of EcSSB protein on the enzymatic activity of TdT in the presence of Me2+: control: 40 nM TdT, 0.2 mM TTP, 5 mM AcOK, 2 mM AsOTris (pH 7.9), 1 mM AcOMg, 0.4 µm oligonucleotide T15 and 2 µm EcSSB.

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11. 10. Chromatograms of T15 elongation products (tR 12.4 min) reflecting the effect of Me2+ and EcSSB cations: control: 40 nM TdT, 0.2 mM TTP, 5 mM AcOK, 2 mM AsOTris (pH 7.9), 1 mM AcOMg, 0.25 mM Me2+, 0.4 µm T15 and 2 µm EcSSB.

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12. 11. Effect of Sso7d on TdT activity in the elongation reaction of T5, T15, T35 under various conditions. The effect of Zn2+ and Co2+ cations on the elongation of T15 is reflected.

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