Characterization and Engineering of Two Novel Strand-Displacing B Family DNA Polymerases from Bacillus Phage SRT01hs and BeachBum
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Plasmid Construction and Protein Expression
2.3. Protein Expression
2.4. Structural Analysis
2.5. Exonuclease Activity
2.6. The Preparation of Single-Strand Circular (SSC) Templates
2.7. Rolling Circle Amplification (RCA)
2.8. DNA Replication Using a Hairpin Linear Template and Unnatural Substrates
2.9. Polymerase Fidelity Analysis
3. Results
3.1. SRHS and BBum DNA Polymerases Exhibit Phi29-Like Strand Displacement, Exonuclease Activity, and Processivity
3.2. Rational Design of SRHS and BBum Polymerase Mutants
3.2.1. Mutants Targeting Exonuclease Activity
3.2.2. Mutagenesis Targeting Processivity and Strand Displacement
3.3. Exonuclease Activity in SRHS and BBum Mutants
3.4. Processivity Analysis of SRHS and BBum Mutants
3.5. Processivity of Mutant Variants Using Unnatural Substrates
3.6. Key Mutantions Enabling Mg2+-Dependent Unnatural Substrate Incorporation
4. Discussion
4.1. Exonuclease Activity and Structural Implications
4.2. Processivity and Strand Displacement Activity
4.3. Processivity with Unnatural Substrates
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mutant Name | Mutant Sites |
---|---|
SRHS_A | K129S |
SRHS_B | K129S, D538A |
SRHS_C | K129S, Q132K, D538K |
SRHS_D | K129S, Q132K, S187R, N196K, D538K |
SRHS_E | M97K, K129S, D486E, K515Y, D538A |
SRHS_F | G96R, M97K, K129S, D486E, K515Y, D538A |
SRHS_G | D486E, K515Y |
SRHS_H | K129S, Q132K |
SRHS_I | M97K, D486E, K515Y |
SRHS_J | K129S, D538K |
SRHS_K | S187R, N196K |
SRHS_L | G96R, M97K, D486E, K515Y, |
SRHS_M | G96R, M97K, S187R, N196K, D486E, K515Y, |
BBum_Exo_A | D12A |
BBum_Exo_B | N63D |
BBum_Exo_C | Y166C |
BBum_Exo_D | D170A |
BBum_Pro_E | D150H, A151K, P152E |
BBum_Pro_F | A493E |
BBum_Pro_G | S97R, M98K, |
BBum_Pro_H | S97R, M98K, A493E |
BBum_Pro_I | S97R, M98K, A493E, (phi29 (507–524) instead of 27(516–542)) |
BBum_Pro_J | S97R, M98K, A493E, (phi29 (501–524) instead of 27(510–542)) |
BBum_Pro_K | S97R, M98K, A493E, (phi29 (507–524, K512Y) instead of 27(516–542)) |
BBum_Pro_L | S97R, M98K, A493E, (phi29 (501–524, K512Y) instead of 27(510–542)) |
BBum_Pro_M | S97R, M98K, A493E, (CPS2 (662–674) instead of 27(510–542)) |
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Sun, Y.; Fu, K.; Lin, W.; Gao, J.; Zhao, X.; He, Y.; Tian, H. Characterization and Engineering of Two Novel Strand-Displacing B Family DNA Polymerases from Bacillus Phage SRT01hs and BeachBum. Biomolecules 2025, 15, 1126. https://doi.org/10.3390/biom15081126
Sun Y, Fu K, Lin W, Gao J, Zhao X, He Y, Tian H. Characterization and Engineering of Two Novel Strand-Displacing B Family DNA Polymerases from Bacillus Phage SRT01hs and BeachBum. Biomolecules. 2025; 15(8):1126. https://doi.org/10.3390/biom15081126
Chicago/Turabian StyleSun, Yaping, Kang Fu, Wu Lin, Jie Gao, Xianhui Zhao, Yun He, and Hui Tian. 2025. "Characterization and Engineering of Two Novel Strand-Displacing B Family DNA Polymerases from Bacillus Phage SRT01hs and BeachBum" Biomolecules 15, no. 8: 1126. https://doi.org/10.3390/biom15081126
APA StyleSun, Y., Fu, K., Lin, W., Gao, J., Zhao, X., He, Y., & Tian, H. (2025). Characterization and Engineering of Two Novel Strand-Displacing B Family DNA Polymerases from Bacillus Phage SRT01hs and BeachBum. Biomolecules, 15(8), 1126. https://doi.org/10.3390/biom15081126