Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD111Edited by R. Huber

Hashimoto, Hiroshi; Nishioka, Motomu; Fujiwara, Shinsuke; Takagi, Masahiro; Imanaka, Tadayuki; Inoue, Tsuyoshi; Kai, Yasushi

doi:10.1006/jmbi.2000.4403

Cited by 144 publications

(112 citation statements)

References 28 publications

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“…8D), the archaea Thermococcus sp. 9°N-7 (32), Pyrococcus kodakaraensis KOD1 (33), Thermococcus gorgonarius (34), Sulfolobus solfataricus (35) (Fig. 8E), and Desulfurococcus tok (36) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family

Hogg

Aller

Konigsberg

et al. 2007

Journal of Biological Chemistry

117

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Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a ␤ hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the ␤ hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the ␤ hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 Å crystal structure of the ␤ hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the ␤ hairpin loop.

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Section: Discussionmentioning

confidence: 99%

Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family

Hogg

Aller

Konigsberg

et al. 2007

Journal of Biological Chemistry

117

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“…Several crystal structures of PolB have been determined, and they share essentially the same architecture, consisting of five domains: finger, palm, thumb (the three polymerase domains), N-term, and 3 0 → 5 0 exonuclease domains (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The exonuclease domain, which is responsible for the 3 0 → 5 0 editing of incorrect synthesis by the polymerizing activity, ensures the high fidelity of DNA replication (11,12).…”

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confidence: 99%

Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

Mayanagi

Kiyonari

Nishida

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the threedimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNAinteracting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.fidelity control | protein-DNA complex | replication fork | single particle analysis | structural bioinformatics

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“…Structural and kinetic analyses of Family A (7)(8)(9)(10)(11)(12)(13)(14) and Family B (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) DNA polymerases have increased the understanding of nucleotide selection and incorporation mechanisms. Although amino acid sequences diverge between these two families, the structures of Family A and B DNA polymerases share recognizable finger, thumb, and palm subdomains that allow comparison of structural elements important for function (3,11).…”

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confidence: 99%

Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

Gardner

Joyce

Jack

2004

Journal of Biological Chemistry

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Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent A488L DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.All free living organisms encode several DNA polymerases that are jointly responsible for the replication and maintenance of their genomes, thereby ensuring accurate transmission of genetic information (1-3). The majority of identified DNA polymerases can be classified into Families A, B, C, and Y according to amino acid sequence similarities to Escherichia coli polymerases I, II, III, and IV/V, respectively (4, 5). Additional families have been identified, including the two-subunit replicative DNA polymerases from hyperthermophilic Archaea (Family D) (6) and eukaryotic DNA polymerase ␤ and terminal transferases (Family X) (4).Structural and kinetic analyses of Family A (7-14) and Family B (15-25) DNA polymerases have increased the understanding of nucleotide selection and incorporation mechanisms. Although amino acid sequences diverge between these two families, the structures of Family A and B DNA polymerases share recognizable finger, thumb, and palm subdomains that allow comparison of structural elements important for function (3, 11). In the case of Family A DNA polymerases from bacteriophage T7, Escherichia coli (Klenow fragment, large fragment of DNA polymerase I), and Thermus aquaticus, as well as the Family B DNA polymerase from bacteriophage RB69, interpretation of the structural information is complemented by steady-state and pre-steady-state kinetic studies, allowing a detailed description of the polymerization pathway. Reaction parameters describing the discrimination against naturally occurring nucleotide analogs encountered in vivo, such as NTPs, or unnatural n...

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Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD111Edited by R. Huber

Cited by 144 publications

References 28 publications

Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family

Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family

Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

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