Characterization and Engineering of Two Novel Strand-Displacing B Family DNA Polymerases from Bacillus Phage SRT01hs and BeachBum

Abstract

Polymerase-coupled nanopore sequencing requires DNA polymerases with strong strand displacement activity and high processivity to sustain continuous signal generation. In this study, we characterized two novel B family DNA polymerases, SRHS and BBum, isolated from Bacillus phages SRT01hs and BeachBum, respectively. Both enzymes exhibited robust strand displacement, 3'→5' exonuclease activity, and maintained processivity under diverse reaction conditions, including across a broad temperature range (10-45 °C) and in the presence of multiple divalent metal cofactors (Mg²⁺, Mn²⁺, Fe²⁺), comparable to the well-characterized Phi29 polymerase. Through biochemical analysis of mutants designed using AlphaFold3-predicted structural models, we identified key residues (G96, M97, D486 in SRHS; S97, M98, A493 in BBum) that modulated exonuclease activity, substrate specificity and metal ion utilization. Engineered variants SRHS_F and BBum_Pro_L efficiently incorporated unnatural nucleotides in the presence of Mg²⁺-a function not observed in Phi29 and other wild-type strand-displacing B family polymerases. These combined biochemical features highlight SRHS and BBum as promising enzymatic scaffolds for nanopore-based long-read sequencing platforms.

Keywords: Mg2+-dependence modified substrate incorporation; Phi29-like processivity; exonuclease activity.

Figure 1

The exonuclease activity and processivity analysis of the SRHS and BBum polymerases. (A) The AlphaFold3-predicted structures of the SRHS (pTM = 0.95) and BBum (pTM = 0.95) polymerases exhibited overall topological similarity to the experimentally determined structure of Phi29 (PDB: [2PYL]). A pTM > 0.95 indicated an expected structural deviation of less than 5% in conserved domains compared to experimental data. The Phi29 structure was color-coded as follows: the exonuclease domain (residues 1–190) in red; palm domain (residues 191–261 and 428–531) in pink; TPR1 domain (residues 262–359) in orange; fingers domain (residues 360–395) in blue; TPR2 domain (residues 396–427) in cyan; and thumb domain (residues 532–575) in green. In the predicted structures, regions with high pLDDT scores (>90) are colored blue, while regions with moderate pLDDT scores (70–90) are colored cyan. (B) The exonuclease activity assays of various polymerases under four oligonucleotide conditions: M25 (unmodified), M25-3′ (with three consecutive phosphorothioate (PS) modification at the 3′ terminal), M25-5′ (with three consecutive PS modifications at the 5′ terminal), and M25-3′5′ (with PS modifications at both terminals). The reactions were conducted using 1 µM oligonucleotides and 200 nM of polymerase in 1× phi29 reaction buffer. The mixtures were incubated at 30 °C for 30 min, followed by analysis using 10% TBE-Urea gel and quantification with Image J. Levels were normalized to the negative control (NC) which just added oligonucleotides. Three constitutive PSs as a protecting group can protect oligonucleotides from degrading. (C) Rolling circle amplification (RCA) assays revealed the high processivity of SRHS, BBum, and Phi29. In total, a 100 nM primed template was incubated with 100 nM polymerases and 100 μM dNTPs in Mg²⁺-containing buffer at 30 °C for 1 h. The result was then analyzed through 0.6% agarose gel electrophoresis. NC refers to negative control which just possessed templates. M1 refers to the 1 KB Plus DNA Ladder; M2 refers to the GeneRuler High Range DNA Ladder.

Figure 2

The biochemical characteristics of the Phi29, SRHS, and BBum polymerases. (A–C) The effect of temperature on the polymerase activity of the Phi29, SRHS, and BBum polymerases. A 100 nM primed template was incubated with 100 nM polymerases and 100 μM dNTPs at different temperatures for 1 h in Mg²⁺-containing reaction buffer. The results were analyzed with 0.6% agarose gel electrophoresis. (D) The effect of different metal ions on the polymerase activity of the Phi29, SRHS, and BBum polymerases. Reactions were performed with a 100 nM primed template, 100 nM polymerase, and 100 μM dNTPs at 30 °C for 1 h in reaction buffers supplemented with different divalent metal ions (1 mM final concentration), and products were analyzed by 0.6% agarose gel electrophoresis. NC refers to negative control which just possessed templates. M1 refers to the 1 KB Plus DNA Ladder; M2 refers to the GeneRuler High Range DNA Ladder.

Figure 3

The mutant sites and structure domain analysis of the Phi29, SRHS, and BBum polymerases. (A) The mutant sites in the SRHS and BBum mutant variants. The red stars refer to the protruding structures near the substrate entrance of DNA polymerases. (B) The sequence alignments of the CPS2, Phi29, SRHS, and BBum polymerases. Green indicates identical amino acids; The red box refers to different lengths of the protruding structures near the substrate entrance in the CPS2, Phi29, SRHS, and BBum polymerases. (C) The structure alignments of the Phi29, SRHS, and BBum polymerases showed a distinctive protruding structure near the substrate entrance in the BBum polymerase compared with the Phi29 and SRHS polymerases.

Figure 4

The exonuclease activity analysis of SRHS and BBum mutant variants. (A,B) Exonuclease activity analysis of polymerase mutant variants. We mixed 1 µM M25-5′ oligonucleotides and 200 nM polymerase mutant variants in a 1 × phi29 buffer at 30 °C for 30 min. The results were analyzed by 10% TBE-Urea gel electrophoresis and quantified by ImageJ (version 2.35; National Institutes of Health, Bethesda, MD, USA). Levels were normalized to the negative control (NC) which just added oligonucleotides.

Figure 5

The processivity analysis of SRHS and BBum mutant variants. (A,B) We incubated a 100 nM primed template with 100 nM polymerases and 100 μM dNTPs for 1 h. The results were then analyzed on 0.6% agarose gel electrophoresis. M1 refers to the 1 KB Plus DNA Ladder; M2 refers to the GeneRuler High Range DNA Ladder. NC refers to the negative control which just added the primed template.

Figure 6

The processivity of SRHS and BBum polymerases with unnatural substrates. (A,B) The effect of metal cofactors on DNA extension. We incubated a 100 nM primed template with 100 nM polymerases and 100 μM unnatural substrates at 30 °C for 1 h in reaction buffers containing different metal ions (1 mM final concentration). The results were then analyzed through 0.6% agarose gel electrophoresis. (C–E) The extension efficiency of mutant variants in unnatural substrate and Mn²⁺ conditions. We incubated a 100 nM primed template with 100 nM polymerases and 100 μM unnatural substrates at 30 °C for 1h in reaction buffers. The results were then analyzed through 0.6% agarose gel electrophoresis. M1 referred to the 1 KB Plus DNA Ladder; M2 referred to the GeneRuler High Range DNA Ladder. NC refers to the negative control which just added the primed template and unnatural substrates.

Figure 7

The processivity of SRHS and BBum polymerases with unnatural substrates and Mg²⁺. (A–C) Extension ability analysis of mutant variants with a hairpin template. The 20 nM hairpin template was incubated with 10 nM polymerases and 100 μM unnatural substrates at 30 °C for 30 min in Mg²⁺ -containing buffer. The results were then analyzed on 10% TBE-Urea gel. M refers to the O’RangeRuler 10 bp DNA Ladder. NC refers to the negative control with templates and unnatural substrates. (D) Extension ability analysis of mutant variants with primed SSC templates. We incubated a 100 nM primed template with 100 nM polymerases, 100 μM unnatural substrates, and 1 mM Mg²⁺ at 30 °C for 1h. The results were then analyzed on 0.6% agarose gel electrophoresis. M1 refers to the 1 KB Plus DNA Ladder. NC refers to the negative control which just added the primed template and unnatural substrates.