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 Glu³⁷⁵ 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.

Fig. 1

Diagram of chimeric Phi29EL Pol. (A) E‐L (HhH)₂ repeats from Topo V (pink) were fused to the C‐terminal end of Phi29 Pol (white) with a linker sequence (GTGSGA) between them. Ls, linker sequence. (B) The 3D structure of Phi29EL Pol modelled by Swiss‐PdbViewer. Phi29 Pol structure (PDB: 2PYL) complexed with substrate DNA (green) was downloaded from PDB. The modular structure of E‐J domains was predicted using SWISS‐MODEL. Both domains are shown in the same colours as described for Panel A. The template/primer junction is shown in green. The COOH termini of Phi29 Pol and NH₂ termini of (HhH)₂ domains are shown in red and cyan, respectively. The linker sequence (Ls) is shown as a blue line. Except for the G motif and part of the F motif being disordered, all the other domains matched well with the crystal structure of the Topo‐97 fragment (PDB: 5HM5). The spatial arrangement of (HhH)₂ domains is beneficial for DNA binding.

Fig. 2

Processivity of Phi29 Pol and Phi29EL Pol with singly primed M13mp18 single‐strand DNA. Processivity was quantified by the gradual dilution of Phi29 Pol (A) and Phi29EL Pol (B). After incubating at 30°C for 30 min, samples were mixed with loading buffer for electrophoresis. The molar concentrations of enzymes were 20‐ (Lane 1), 10‐ (Lane 2), 5‐ (Lane 3) and 2.5‐fold (Lane 4) more than that of the M13 template. (C) Quantification of newly synthesized DNA from rolling circle replication. Data are shown as product concentrations versus molar ratios of DNA polymerase to M13 ssDNA. The dotted line and solid line represent Phi29 Pol and Phi29EL Pol, respectively. DNA length markers are labelled with arrowheads in A and B.

Fig. 3

Salt tolerance of Phi29 Pol and Phi29EL Pol. Divergent salts ranging from 0 to 1 M were separately added into the hairpin extension reactions with Phi29 Pol (A) and Phi29EL Pol (B). TBE‐urea gels (20%) were used to separate the templates and extension products. Complete extension products in A and B were quantified by band intensity. The y‐axis in panels C and D indicates the product concentration relative to the total amount of template in the last lane of each gel. Data are presented as means ± SD. (E) The hairpin sequence was 53 nt, while the extension product was 74 nt.

Fig. 4

Salt tolerance of Phi29 Pol and Phi29EL Pol. Different concentrations of NaCl or KCl were added into the singly primed M13mp18 extension experiment. Products of Phi29 Pol (A) and Phi29EL Pol (B) were separated using 0.6% alkaline agarose gels. Lane 1, background reaction mixtures without enzyme; Lane 2, no added salt; Lanes 3–5, 0.1, 0.2 and 0.3 M NaCl; Lanes 6–8 with 0.1, 0.2 and 0.3 M KCl. The yielded DNA (> 48 kb) was further quantified in C (NaCl) and D (KCl). Data are shown as product amounts versus salt concentrations. Data are presented as means ± SD.

Fig. 5

Salt tolerance of KAc and KGlc for Phi29 Pol (A) and Phi29EL Pol (B). KAc or KGlc at 0–0.3 M added to the reaction mixture separately with singly primed M13mp18 as templates. Lane 1, background reaction without enzyme; Lane 2, no added salt; Lanes 3–5, with 0.1, 0.2 or 0.3 M KAc; Lanes 6–8 with 0.1, 0.2 or 0.3 M KGlc. Quantification analysis of the synthesized DNA under increased concentrations of KAc (C) and KGlc (D). Compared to NaCl and KCl, both of the enzymes were much more resistant to KAc and sensitive to KGlc. Data are presented as means ± SD.

Fig. 6

Exonuclease activity of Phi29 Pol and Phi29EL Pol. A. Sequences and modifications of Oligos A, B and C. The lowercase ‘s’ denotes phosphorothioate. B. Both polymerases (200 nM) were separately mixed with three oligos for 10 min. The reactions were analysed using a 20% TBE‐urea gel. 3′‐5′ proofreading exonuclease activity of both enzymes could degrade natural oligos (Oligo A) and 5′ phosphorothioate‐modified oligos (Oligo C) after a short time of incubation. C. Specific activity assay. A 3′ to 5′ exonuclease activity assay was performed with Phi29 Pol or Phi29EL Pol according to the manufacturer’s instructions. Data are presented as means ± SD.

Fig. 7

Salt tolerance of Phi29EL Pol mutants. KCl at 0 M or 0.3 M was separately added into the M13 extension system. The reaction time was 30 min. DNA length markers are labelled with arrowheads. Data are representative of several independent experiments.