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

Abstract

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.

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase

“…The appropriate concentration of salt ions in the enzyme storage buffer plays a key role in the activity and stability of the enzyme (Gao et al, 2021). Salt stress caused by high salt concentration will affect the structural integrity of the polymerase, hindering the anchoring of DNA on the polymerase, and replication initiation (Pessôa Filho et al, 2011).…”

“…The loop of the TP Regions (TPR1) subdomain of phi29 DNA polymerase was studied by changing several residues into alanine, revealing primer-terminus stabilization at the polymerization active site (del Prado et al, 2019). A chimeric phi29 DNA polymerase with helix-hairpin-helix motifs was created in order to enhance salt tolerance and replication performances (Gao et al, 2021). DNA binding ability was improved by the fusion of a Helix-hairpin-Helix domain without affecting the replication rate (de Vega et al, 2010).…”

Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform

“…With respect to stability and conservative analysis, Y369E, T372N, E375E, and I378R all exhibited better conservative traits (Figure 3A), whereas Y369K, T372E, E375Q, and I378L exhibited the highest frequency, and Y369K (88), T372Q (100), E375N (100), and I378R (100) scored the highest in terms of stability according to the HotSpot Wizard. Asn, the amino acid with the highest frequency at position 375, has been reported to possess elevated salt tolerance (Gao et al, 2021). Although both Gln and Asn exhibited a similar charge characteristic, it is worth noting that both Asp and Asn have shorter side chains than Glu and Gln.…”

“…we mutated Y369K to SZ_B. Ser at position 375 of phi29 has been reported to possess elevated salt tolerance (Gao et al, 2021), and Asn and Gln have similar charge characteristics at position 372. Hence, the addition of E375S to SZ_C and E375S and T372Q to SZ_D were studied to evaluate their respective functions.…”

“…Different HhH motifs may have different effects on extension rates. When the H and I motifs from the [(HhH)2] domain were coupled with phi29 polymerase, its DNA-binding affinity and salt tolerance were drastically enhanced (de Vega et al, 2010;Gao et al, 2021). However, the addition of a C-terminal domain results in a longer linker between the polymerase and the nanopore, thus hindering the capture of an electrochemical signal.…”