Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen from<i>Thermococcus kodakaraensis</i>KOD1

Kitabayashi, Masao; Nishiya, Yoshiaki; Esaka, Muneharu; Itakura, Mitsuo; Imanaka, Tadayuki

doi:10.1271/bbb.66.2194

Cited by 14 publications

(11 citation statements)

References 28 publications

(21 reference statements)

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“…Stimulation of archaeal DNA synthesis by PCNA on a circular DNA template was observed without the clamp-loader. 21,29,30 This was attributed to spontaneous loading that could be explained by weaker intermolecular interactions within PCNA monomers. 31 Moreover, the residues that maintain the toroidal structure of the archaeal PCNA were identified.…”

Section: Introductionmentioning

confidence: 99%

DNA Polymerase Switching on Homotrimeric PCNA at the Replication Fork of the Euryarchaea Pyrococcus abyssi

Rouillon

Henneke

Flament

et al. 2007

Journal of Molecular Biology

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DNA replication in Archaea, as in other organisms, involves large protein complexes called replisomes. In the Euryarchaeota subdomain, only two putative replicases have been identified, and their roles in leading and lagging strand DNA synthesis are still poorly understood. In this study, we focused on the coupling of proliferating cell nuclear antigen (PCNA)-loading mechanisms with DNA polymerase function in the Euryarchaea Pyrococcus abyssi. PCNA spontaneously loaded onto primed DNA, and replication factor C dramatically increased this loading. Surprisingly, the family B DNA polymerase (Pol B) also increased PCNA loading, probably by stabilizing the clamp on primed DNA via an essential motif. In contrast, on an RNA-primed DNA template, the PCNA/Pol B complex was destabilized in the presence of dNTPs, allowing the family D DNA polymerase (Pol D) to perform RNA-primed DNA synthesis. Then, Pol D is displaced by Pol B to perform processive DNA synthesis, at least on the leading strand.

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Section: Introductionmentioning

confidence: 99%

DNA Polymerase Switching on Homotrimeric PCNA at the Replication Fork of the Euryarchaea Pyrococcus abyssi

Rouillon

Henneke

Flament

et al. 2007

Journal of Molecular Biology

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“…25,26 Interesting, in the four hyperthermophilic archaea investigated so far (Pyrococcus furiosus, Archaeoglobus fulgidus, Pyrococcus abyssi and Thermococcus kodakaraensis), the RFC does not require ATP hydrolysis for the clamp loading, unlike bacterial and eukaryal clamp loaders. 27,28,20,30 Even more interesting is the fact that PCNA is apparently able to load itself in the absence of RFC, even on circular DNA templates. It is suggested that spontaneous loading of hyperthermophilic archaeal PCNA may be facilitated by the high temperatures at which those organisms live, and the function of RFC may be to unload PCNA rather than to load it.…”

Section: Replication In Archaea Bacteria and Eukaryamentioning

confidence: 99%

Overview of Archaea

Pikuta

2011

SPIE Proceedings

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Archaea were separated from Eubacteria after discovery of their specifics in cell outer membrane that usually not affected by common antibiotics. Phylogenetic analysis introduced by Karl Wöese supported this separation. Presently, only two phyla Crenarchaeota and Euryarchaeota include the valid representatives. Another three phyla that were proposed based on the sequence analysis of environmental samples, do not contain validly published species, and for this reason they are not included in this review. The phylum Euryarchaeota currently includes eight classes and ten orders, while the Crenarchaeota phylum contains the only class with five orders. Members of the phyla Crenarchaeota have two or three family B and no family D DNA polymerases, but members of the Euryarchaeota contain the only family B polymerases and the only family D polymerases, and it is still not clear, which is the main functional enzyme in the replication process.In this article, we are present an update and comparative analysis for this domain, discussing unique features of this group and Evolution, estimating their physiology within the matrix of physic-chemical factors, and outlining future perspectives in their study. Rules of the diagonal for the diagrams with all Archaea are presented and discussed.

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“…However, in vitro , e.g., during the PCR, PCNA is not usually present and copying of DNA relies on the intrinsic processivity of the polymerase. Occasionally PCNA has been added and has been reported to enhance the PCR capability of Tkod-Pol (Kitabayashi et al, 2002). A recent comprehensive study showed that native Pfu-PCNA only improved PCR if RFC was also present, presumably to facilitate loading of the clamp onto DNA (Ishino et al, 2012).…”

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 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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Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen fromThermococcus kodakaraensisKOD1

Cited by 14 publications

References 28 publications

DNA Polymerase Switching on Homotrimeric PCNA at the Replication Fork of the Euryarchaea Pyrococcus abyssi

DNA Polymerase Switching on Homotrimeric PCNA at the Replication Fork of the Euryarchaea Pyrococcus abyssi

Overview of Archaea

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

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