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

Abstract

In the current work, single strand binding protein from E. coli (EcSSB) and DNA-binding protein from S. solfataricus (Sso7d) were tested to evaluate the effects on TdT activity for homopolymer substrates (T_n) that are unable to form double helix structures. We showed a significant increase in TdT activity after the addition of EcSSB even from the example of homopolymer substrates. The 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 in 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.