Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon <i>Pyrococcus abyssi</i>

Gueguen, Yannick; Rolland, Jean‐Luc; Lecompte, Odile; Azam, Philippe; Romancer, Gisèle Le; Flament, Didier; Raffin, Jean-Paul; Diétrich, Jacques

doi:10.1046/j.0014-2956.2001.02550.x

Cited by 60 publications

(77 citation statements)

References 36 publications

(62 reference statements)

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“…It was found that DP1 is conserved at its middle and COOH-terminal (from around 201 to 622), but not at its NH 2 -terminal. The most conserved region is from residue 300 to 600, which contains a sequence homologous to the small subunit of eukaryotes DNA polymerase ␦ (19) and a putative phosphoesterase domain for the 3Ј-5Ј exonuclease (18,21). The conserved regions of DP2 are located at residues 1-300, 350 -500, 650 -750, and 800 -1434.…”

Section: Resultsmentioning

confidence: 99%

“…NRC01 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Some members of the DNA polymerase family have been cloned and biochemically characterized, such as PolDs from P. furiosus (15), M. jannaschii (16), P. horikoshii (17), and P. abyssi (18), nevertheless, no structural information on PolD is available at present.…”

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confidence: 99%

“…In the genomes of the genus Pyrococcus, the genes coding the two subunits of PolD are adjacent to the replication origin and several essential genes for DNA metabolism (20). DP1 shows low but significant homology to the small subunit of eukaryote DNA polymerase ␣, ␦, and ⑀ (19, 21), and contains domain and catalytic motifs for the 3Ј-5Ј exonuclease, similar to those of Mre11, a nuclease involved in double strand DNA break repair (18,21,22). The amino acid sequences of DP2 contain a short region at the COOH-terminal, which shows homology to the catalytic subunit of yeast DNA polymerase ⑀ (this paper) around the COOH-terminal putative zinc finger motif (cysteine cluster II).…”

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confidence: 99%

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Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

Shen

Tang

Matsui

2003

Journal of Biological Chemistry

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We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cysteine cluster II) of DP2Pho at the COOH-terminal. We found that the internal region of the zinc finger motif is critical for the 3-5 exonuclease, but is dispensable for the DNA polymerization.Family D DNA polymerase (PolD) 1 is a recently found DNA polymerase that exists extensively in Euryarchaeota of Archaea, the third domain of life (1, 2 PolD is composed of a small subunit (DP1) and a large subunit (DP2). The interaction of DP1 and DP2 is essential for the stability and full activity of the enzyme (15, 16). DP1 or DP2 expressed in Escherichia coli is unstable and easily degraded. PolD possesses strong DNA polymerizing and 3Ј-5Ј exonuclease (proofreading) activities like other DNA replication polymerases, however, common motifs for the catalysis of other DNA polymerases are absent or not functional in PolD (17,19). Two invariant residues, Asp 1122 and Asp 1124 of DP2Pho, located in the most conserved region in the large subunit of PolD, were identified to be the catalytic residues for the DNA polymerization activities (17), indicating that the COOH-terminal of DP2 is involved in the formation of the catalytic center for DNA synthesis. A heterotetrameric structure for PolD from P. horikoshii (PolDPho) was proposed based on the result of gel filtration (17).Several lines of evidence indicate that family D DNA polymerase is likely the major DNA polymerase or replicase in Euryarchaeota, participating in DNA replication, repair, and recombination. In the genomes of the genus Pyrococcus, the genes coding the two subunits of PolD are adjacent to the replication origin and several essential genes for DNA metabolism (20). DP1 shows low but significant homology to the small subunit of eukaryote DNA polymerase ␣, ␦, and ⑀ (19, 21), and contains domain and catalytic motifs for the 3Ј-5Ј exonuclease, similar to those of Mre11, a nuclease involved in double strand DNA break repair (18,21,22). The amino acid sequences of DP2 contain a short region at the COOH-terminal, which shows homology to the catalytic subunit of yeast DNA polymerase ⑀ (this paper) around the COOH-terminal putative zinc finger motif (cysteine cluster II). This homologous region in yeast polymerase ⑀ is found to be vital for the survival of yeast (23,24). PolD interacts with many proteins related to DNA replication, repair, and recombination. It was found that DP1 from P. furiosus interacts specifically with archaeal RadB by yeast two-hybrid analysis and in vitro pull-down assays (25). DP2 directly interacts with proliferating cell nuclear antigen as * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Section: Resultsmentioning

confidence: 99%

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Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

Shen

Tang

Matsui

2003

Journal of Biological Chemistry

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“…The enzyme is composed of a small subunit (DP1) with 3=¡5= exonuclease activity and a large subunit (DP2) that contains the polymerase activity. Individually, the subunits have low activity, but when assembled into a heterodimer, or further into a heterotetramer, both activities are substantially increased (14,15,(25)(26)(27). In vitro, Pol D behaves as a template-dependent DNA polymerase, has proofreading activity, and is capable of strand displacement DNA synthesis (13, 28).…”

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Archaeal DNA Polymerase D but Not DNA Polymerase B Is Required for Genome Replication in Thermococcus kodakarensis

et al. 2013

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c Three evolutionarily distinct families of replicative DNA polymerases, designated polymerase B (Pol B), Pol C, and Pol D, have been identified. Members of the Pol B family are present in all three domains of life, whereas Pol C exists only in Bacteria and Pol D exists only in Archaea. Pol B enzymes replicate eukaryotic chromosomal DNA, and as members of the Pol B family are present in all Archaea, it has been assumed that Pol B enzymes also replicate archaeal genomes. Here we report the construction of Thermococcus kodakarensis strains with mutations that delete or inactivate key functions of Pol B. T. kodakarensis strains lacking Pol B had no detectable loss in viability and no growth defects or changes in spontaneous mutation frequency but had increased sensitivity to UV irradiation. In contrast, we were unable to introduce mutations that inactivated either of the genes encoding the two subunits of Pol D. The results reported establish that Pol D is sufficient for viability and genome replication in T. kodakarensis and argue that Pol D rather than Pol B is likely the replicative DNA polymerase in this archaeon. The majority of Archaea contain Pol D, and, as discussed, if Pol D is the predominant replicative polymerase in Archaea, this profoundly impacts hypotheses for the origin(s), evolution, and distribution of the different DNA replication enzymes and systems now employed in the three domains of life. DNA replication, an essential event for all cellular life, is catalyzed by protein complexes designated replisomes, in which individual activities are tightly regulated and coordinated. DNA polymerases are the functional center of the replisome, but structurally distinct DNA polymerases, designated family C (Pol C) and family B (Pol B) polymerases, catalyze genome replication in Bacteria and eukaryotes, respectively (1-3). This difference has led to much debate, most fundamentally regarding whether DNA replication has evolved more than once, possibly independently in different biological lineages (1, 4-10). All known archaeal genomes encode at least one member of the Pol B family, and given that Archaea are evolutionarily closer to eukaryotes than are Bacteria (11, 12), it has been tacitly assumed, but challenged (13,14), that Pol B enzymes must also replicate archaeal genomes. Presumably, this must be the case for the Crenarchaeota, as their genomes appear to encode only Pol B enzymes. This is, however, only an assumption for all members of the Euryarchaeota, Thaumarchaeota, Korarchaeota, Aigarchaeota, and Nanoarchaeota lineages, as their genomes encode not only Pol B enzymes but also members of an archaeon-specific DNA polymerase family designated Pol D (Fig. 1) (11, 13-15).The Thermococcales are hyperthermophilic Euryarchaea, and given the commercial value of thermostable processive DNA polymerases, Pol B polymerases from this genus have received extensive characterization (13,16,17). Within these single polypeptide enzymes, the regions and residues directly responsible for nucleotide polymerization, 3=¡...

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“…Two DNA polymerase (Pol I and II) that exhibit a 3′→5′ exonuclease proofreading activity were also isolated from Pyrococcus abyssi (Gueguen et al 2001). Pol I DNA polymerase from P. abyssi (Pab), marketed by Qbiogen as "Isis DNA Polymerase", has an error rate (0.66×10 −6 ) similar to that of Pfu.…”

Section: Tth Polymerasementioning

confidence: 99%

Enzymes used in molecular biology: a useful guide

Rittié

Perbal

2008

Journal of Cell Communication and Signaling

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Since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. Thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. This review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. We provide a description of the most commonly used enzymes of each group, and explain their properties and mechanism of action. By pointing out key requirements for each enzymatic activity and clarifying their limitations, we aim at guiding the reader in selecting appropriate enzymatic source and optimal experimental conditions for molecular cloning experiments.

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Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi

Cited by 60 publications

References 36 publications

Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

Archaeal DNA Polymerase D but Not DNA Polymerase B Is Required for Genome Replication in Thermococcus kodakarensis

Enzymes used in molecular biology: a useful guide

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