Chimeric Phi29 DNA polymerase with helix–hairpin–helix motifs shows enhanced salt tolerance and replication performance

Summary

Phi29 DNA polymerase (Phi29 Pol) has been successfully applied in DNA nanoball‐based sequencing, real‐time DNA sequencing from single polymerase molecules and nanopore sequencing employing the sequencing by synthesis (SBS) method. Among these, polymerase‐assisted nanopore sequencing technology analyses nucleotide sequences as a function of changes in electrical current. This ionic, current‐based sequencing technology requires polymerases to perform replication at high salt concentrations, for example 0.3 M KCl. Nonetheless, the salt tolerance of wild‐type Phi29 Pol is relatively low. Here, we fused helix–hairpin–helix (HhH) ₂ domains E‐L (eight repeats in total) of topoisomerase V (Topo V) from the hyperthermophile Methanopyrus kandleri to the Phi29 Pol COOH terminus, designated Phi29EL DNA polymerase (Phi29EL Pol). Domain fusion increased the overall enzyme replication efficiency by fourfold. Phi29EL Pol catalysed rolling circle replication in a broader range of salt concentrations than did Phi29 Pol, extending the KCl concentration range for activity up to 0.3 M. In addition, the mutation of Glu ³⁷⁵ to Ser or Gln increased Phi29EL Pol activity in the presence of KCl. In this work, we produced a salt‐tolerant Phi29 Pol derivative by means of (HhH) ₂ domain insertion. The multiple advantages of this insertion make it a good substitute for Phi29 Pol, especially for use in nanopore sequencing or other circumstances that require high salt concentrations.

Abstract

1. For ionic current based nanopore sequencing, polymerases, helicases or exonucleases were required to process DNA with relatively higher contents of salt (0.2–1 M KCl) which is necessary to attain sufficient ionic strength for nucleobase recognition and increase the signal to noise ratio. The delicate structure features endow Phi29 Pol intrinsic strand displacement capability, making it an ideal enzyme for various sequencing technologies, for example real‐time DNA sequencing from single polymerase molecules, DNA nanoball‐based sequencers and nanopore sequencing technology employing sequencing by synthesis method. 2. We have achieved a salt tolerant Phi29 Pol derivative working at 0.3 M KCl by means of helix‐hairpin‐helix domain insertion. The enzyme also showed improved rolling circle replication efficiency. 3. Besides salt tolerance, other properties of Phi29EL Pol such as the processivity, strand displacement and extension rate were not affected much by domain fusion. The combined advantages of Phi29EL Pol makes it a good substitute of Phi29 Pol, especially for nanopore sequencing or other circumstances depend on higher contents of salt.