Pyrococcus Furiosus DNA Polymerase Research Articles

The thumb subdomain of Pyrococcus furiosus DNA polymerase is responsible for deoxyuracil binding, hydrolysis and polymerization of nucleotides.

B-family DNA polymerases, which are found in eukaryotes, archaea, viruses, and some bacteria, participate in DNA replication and repair. Starting from the N-terminus of archaeal and bacterial B-family DNA polymerases, three domains include the N-terminal, exonuclease, and polymerase domains. The N-terminal domain of the archaeal B-family DNA polymerase has a conserved deoxyuracil-binding pocket for specially binding the deoxyuracil base on DNA. The exonuclease domain is responsible for removing the mismatched base pair. The polymerase domain is the core functional domain and takes a highly conserved structure composed of fingers, palm and thumb subdomains. Previous studies have demonstrated that the thumb subdomain mainly functions as a DNA-binding element and has coordination with the exonuclease domain and palm subdomain. To further elucidate the possible functions of the thumb subdomain of archaeal B-family DNA polymerases, the thumb subdomain of Pyrococcus furiosus DNA polymerase was mutated, and the effects on three activities were characterized. Our results demonstrate that the thumb subdomain participates in the three activities of archaeal B-family DNA polymerases as a common structural element. Both the N-terminal deoxyuracil-binding pocket and thumb subdomain are critical for deoxyuracil binding. Moreover, the thumb subdomain assists DNA polymerization and hydrolysis reactions, but it does not contribute to the fidelity of DNA polymerization.

Variable termination sites of DNA polymerases encountering a DNA-protein cross-link.

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4–5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.

ＤＮＡクランプ／複製ＤＮＡポリメラーゼ複合体におけるＤＮＡ合成・修正スイッチング機構

Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with a cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction. Electron microscopy analyses confirmed that this complex structure exists among the multiple functional configurations in solution. Together with data from mutational analyses, this structural study indicated that the novel interaction between a stretched loop of PCNA and the PfuPol Thumb domain is quite important, in addition to the authentic PCNA-polymerase recognition site (PIP box). A comparison of the present structures with the previously reported structures of polymerases complexed with DNA suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by stabilizing only the polymerase mode. This proposed mechanism of fidelity control of replicative DNA polymerases was supported by experiments, in which a mutation within the second interaction site caused an enhancement in the exonuclease activity in the presence of PCNA.

Characterization of Molecular Mechanisms Underlying Mutations in Dystrophic Epidermolysis Bullosa Using Site-directed Mutagenesis

Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.

Mutant DNA Polymerase for Improved Detection of Single‐Nucleotide Variations in Microarrayed Primer Extension

Mutant detects mutation. A mutant DNA polymerase, which was derived from Pyrococcus furiosus DNA polymerase (M2 in figure), was found to have improved single-nucleotide discrimination properties, and was used for selective microarrayed-primer extensions. This approach allowed multiplexed SNP detection.

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes: One-step purification of Pfu DNA polymerase

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase ( Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase ‘structure-guided’ combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding ‘lead’ ligand X–Trz–Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The ‘lead’ ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand ( oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid ( oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant ( K D) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

DNA replication in thermophiles.

DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.

Functional interdependence of DNA polymerizing and 3'-->5' exonucleolytic activities in Pyrococcus furiosus DNA polymerase I.

Pyrococcus furiosus DNA polymerase I (Pol BI) belongs to the family B (alpha-like) DNA polymerases and has a strong 3'-->5' exonucleolytic activity, in addition to its DNA polymerizing activity. To understand the relationship between the structure and function of this DNA polymerase, three deletion mutants, Delta1 (DeltaLeu746-Ser775), Delta2 (DeltaLeu717-Ser775) and Delta3 (DeltaHis672-Ser775), and two substituted mutants of Asp405, D405A and D405E, were constructed. These substitutions affected both the DNA polymerizing and the 3'-->5' exonucleolytic activities. The Delta1 mutant protein had DNA polymerizing activity with higher specific activity than that of the wild-type Pol BI, but retained only 10% of the exonucleolytic activity of the wild-type. The other two deletion mutants lost most of both activities. These results suggest that the DNA polymerizing and exonucleolytic activities are closely related to each other in the folded structure of this DNA polymerase, as proposed in the family B DNA polymerases.

Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA.

5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.

Low Incorporation of dUMP by Some Thermostable DNA Polymerases May Limit Their Use in PCR Amplifications

Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.