Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: Cloning, characterization and PCR application

Ali, Syed Farhat; Rashid, Naeem; Imanaka, Tadayuki; Akhtar, Muhammad

doi:10.1016/j.jbiosc.2011.03.018

Cited by 10 publications

(4 citation statements)

References 27 publications

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“…polD nucleotide selectivity is relatively low compared with other families, especially for a polymerase implicated in playing a replicative role, with an average nucleotide selectivity of 4.3 × 10 3 . Likewise, the data obtained here correspond well to previously published fidelity data, which suggests that polD has a higher error rate than typical replicative polymerases ( 41 ). We suspect the low fidelity observed for 9°N polD is due to the slow rate of polymerization observed for correct nucleotide incorporation, k pol between 1.8 and 3.1 s −1 , in conjunction with the small decrease observed in k pol from correct to incorrect nucleotide incorporation (from 1.5- to 45-fold).…”

Section: Discussionsupporting

confidence: 90%

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Schermerhorn

Gardner

2015

Journal of Biological Chemistry

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Background: Family D DNA polymerase (polD) is important for replication in most archaea, excluding Crenarchaeota.Results: We report a detailed kinetic characterization of polD nucleotide incorporation, mismatch discrimination, and 3′-5′ exonuclease hydrolysis.Conclusion: Despite evolutionary divergence, polD kinetic pathways share similarities to other DNA polymerase families.Significance: This work contributes to unifying our understanding of DNA polymerase function.

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Section: Discussionsupporting

confidence: 90%

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Schermerhorn

Gardner

2015

Journal of Biological Chemistry

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“…High heat resistance is required for PCR enzymes, and only the DNA polymerases from extreme thermophiles or hyperthermophiles, but not moderate thermophiles, can be used for this purpose. There are many commercial DNA polymerases from Thermococcales in Euryarchaeota, but no crenarchaeal DNA polymerase has been practically used for PCR so far, although several enzymes from Pyrobaculum and Ignicoccus are reportedly applicable (Kähler and Antranikian, 2000 ; Ali et al, 2011 ; Seo et al, 2014 ). The heat-stability of ApePolB3 is actually sufficient for applications to PCR, and this enzyme is more tolerant to salt and heparin.…”

Section: Discussionmentioning

confidence: 99%

Two Family B DNA Polymerases From Aeropyrum pernix, Based on Revised Translational Frames

Daimon

Ishino

Imai

et al. 2018

Front. Mol. Biosci.

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Living organisms are divided into three domains, Bacteria, Eukarya, and Archaea. Comparative studies in the three domains have provided useful information to understand the evolution of the DNA replication machinery. DNA polymerase is the central enzyme of DNA replication. The presence of multiple family B DNA polymerases is unique in Crenarchaeota, as compared with other archaeal phyla, which have a single enzyme each for family B (PolB) and family D (PolD). We analyzed PolB1 and PolB3 in the hyperthermophilic crenarchaeon, Aeropyrum pernix, and found that they are larger proteins than those predicted from the coding regions in our previous study and from public database annotations. The recombinant larger PolBs exhibited the same DNA polymerase activities as previously reported. However, the larger PolB3 showed remarkably higher thermostability, which made this enzyme applicable to PCR. In addition, the high tolerance to salt and heparin suggests that PolB3 will be useful for amplification from the samples with contaminants, and therefore it has a great potential for diagnostic use in the medical and environmental field.

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“…Screening for crystallization conditions was conducted by the hanging-drop vapour-diffusion method using Pc-polymerase which had been expressed and purified as described by Ali et al (2011). The enzyme, at a concentration of 15 mg ml À1 in 20 mM Tris pH 8.2, was dispensed using a Mosquito crystal screening robot (TTP Labtech, Hertfordshire, England) into 96-well SBS flat-bottom plates (Molecular Dimensions, Suffolk, England).…”

Section: Crystallizationmentioning

confidence: 99%

“…The cloning of the gene encoding the family B DNA polymerase from P. calidifontis into the Escherichia coli expression vector pET-21a has been reported previously (Ali et al, 2011). Studies of the purified enzyme (Pc-polymerase), which consists of 783 amino acids, established that its activity is magnesium-ion dependent, with an optimal pH of 8.5, an optimal temperature of 75 C and a half-life of 4.5 h at 95 C. Thus, the enzyme has greater thermostability than the widely used Taq DNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

Guo¹,

Zhang²,

Coker³

et al. 2017

Acta Cryst Sect D Struct Biol

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The family B DNA polymerase from Pyrobaculum calidifontis (Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75 C and a half-life of 4.5 h at 95 C, giving it greater thermostability than the widely used Taq DNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3 0 -5 0 exonuclease activity, as do the related high-fidelity DNA polymerases from Pyrococcus furiosus, Thermococcus kodakarensis KOD1 and Thermococcus gorgonarius, which have extensive commercial applications. Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that the P. calidifontis enzyme is distinct. Here, the structure determination of Pc-polymerase is reported, which has been refined to an R factor of 24.47% and an R free of 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6 and moves by 10.0 Å . The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.

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Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: Cloning, characterization and PCR application

Cited by 10 publications

References 27 publications

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Two Family B DNA Polymerases From Aeropyrum pernix, Based on Revised Translational Frames

Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

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