Structures of KOD and 9°N DNA Polymerases Complexed with Primer Template Duplex

Bergen, K.; Betz, Karin; Welte, Wolfram; Diederichs, Kay; Marx, Andreas

doi:10.1002/cbic.201300175

Cited by 50 publications

(83 citation statements)

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“…The family-B DNA polymerases from the Thermococcales order of the archaea are especially favored for PCR, due to their extreme stability at elevated temperatures and the presence of fidelity-conferring 3′-5′ exonuclease activity (Lundberg et al, 1991; Cline et al, 1996; Takagi et al, 1997; Nishioka et al, 2001). Both the amino acid sequences and X-ray structures of these polymerases demonstrate a high degree of similarity, with structures available for the enzymes isolated from Thermococcus gorgonarius (Tgo-Pol) (Hopfner et al, 1999; Firbank et al, 2008; Killelea et al, 2010), Thermococcus kodakarensis (Tkod-Pol) (Hashimoto et al, 2001; Kuroita et al, 2005; Bergen et al, 2013), Thermococcus species 9°N-7 (9°N-Pol) (Chapin-Rodriguez et al, 2000), Pyrococcus furiosus (Pfu-Pol) (Kim et al, 2008) and Pyrococcus abysii (Pab-Pol) (Gougel et al, 2012). Despite similarities of amino acid sequence and structure, these polymerases have diverse kinetic properties; a strong influence on PCR performance.…”

Section: Introductionmentioning

confidence: 75%

“…However, superimposition of the structures of the two polymerases (Figure 1C) shows that R381 in Tkod-Pol and L381 in Pfu-Pol are spatially equivalent, suggesting the alternative alignment shown in Figure 1B (Hashimoto et al, 2001). Another region that may contribute to processivity is the thumb domain, responsible for binding double-stranded DNA (Firbank et al, 2008; Killelea et al, 2010; Gougel et al, 2012; Bergen et al, 2013). This domain grips DNA tightly and interacts with newly synthesized double-strands, implying an important role in DNA translocation.…”

Section: Introductionmentioning

confidence: 99%

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DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

et al. 2014

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The polymerase chain reaction (PCR) is widely applied across the biosciences, with archaeal Family-B DNA polymerases being preferred, due to their high thermostability and fidelity. The enzyme from Pyrococcus furiosus (Pfu-Pol) is more frequently used than the similar protein from Thermococcus kodakarensis (Tkod-Pol), despite the latter having better PCR performance. Here the two polymerases have been comprehensively compared, confirming that Tkod-Pol: (1) extends primer-templates more rapidly; (2) has higher processivity; (3) demonstrates superior performance in normal and real time PCR. However, Tkod-Pol is less thermostable than Pfu-Pol and both enzymes have equal fidelities. To understand the favorable properties of Tkod-Pol, hybrid proteins have been prepared. Single, double and triple mutations were used to site arginines, present at the “forked-point” (the junction of the exonuclease and polymerase channels) of Tkod-Pol, at the corresponding locations in Pfu-Pol, slightly improving PCR performance. The Pfu-Pol thumb domain, responsible for double-stranded DNA binding, has been entirely replaced with that from Tkod-Pol, again giving better PCR properties. Combining the “forked-point” and thumb swap mutations resulted in a marked increase in PCR capability, maintenance of high fidelity and retention of the superior thermostability associated with Pfu-Pol. However, even the arginine/thumb swap mutant falls short of Tkod-Pol in PCR, suggesting further improvement within the Pfu-Pol framework is attainable. The significance of this work is the observation that improvements in PCR performance are easily attainable by blending elements from closely related archaeal polymerases, an approach that may, in future, be extended by using more polymerases from these organisms.

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Section: Introductionmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

et al. 2014

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“…The structural coordinates for KOD DNA polymerase complexed with duplex DNA were obtained from the crystal structure PDB ID: 4K8Z [36] (with resolution of 2.29 Å). KOD-dTTP model was built based on the 1IG9 coordinates using Prime module (a homology modelling tool) of Schrödinger suite, as there was no structure available with the incoming triphosphates for KOD DNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

et al. 2014

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Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.

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“…Both Euryarchaeota and Crenarchaeota encode family B DNA pols. Family B DNA pols share similar structures to the phage RB69 DNA pol (Wang et al 1997) as demonstrated for the structures of the family B archaeal DNA pols from Thermococcus gorganarius (Tgo) (Firbank et al 2008;Hopfner et al 1999;Killelea et al 2010), Thermococcus kodakarensis (KOD1) (Bergen et al 2013;Hashimoto et al 2001;Kuroita et al 2005), Thermococcus species 9°N-7 (9°N-7) (Rodriguez et al 2000), Pyrococcus furiosus (Pfu) (Kim et al 2008), and Pyrococcus abysii (Pab) (Gouge et al 2012), there are five protein domains. The structure for the Tgo DNA pol, which was the first solved structure for an archaeal DNA pol, is shown in Fig.…”

Section: Introductionmentioning

confidence: 96%

Archaeal DNA polymerases in biotechnology

Zhang

Kang

et al. 2015

Appl Microbiol Biotechnol

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DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

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Structures of KOD and 9°N DNA Polymerases Complexed with Primer Template Duplex

Cited by 50 publications

References 65 publications

DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

Archaeal DNA polymerases in biotechnology

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