Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 Å resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA-protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this ''closed'' conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.Propagation of cells requires faithful DNA replication. This is performed in vivo by DNA polymerases (pols), which attach the appropriate dNTP to the nascent DNA primer strand to match its paired template. Different families of pols are involved in different DNA polymerization processes including not only DNA replication (1, 2) but also repair and recombination (3, 4), a heterogeneity also reflected by varying polypeptide structures and͞or subunit compositions (3, 5). Some pols complement polymerase activity with 3Ј 3 5Ј exonuclease activity (editing activity) and͞or 5Ј 3 3Ј ''structure-specific endonuclease'' activity, often located in separate structural domains on the same polypeptide chain (4-8).Crystal structures are available for most known polymerase families, including the A family DNA polymerases (9-14), pol ␤ (15-17), HIV reverse transcriptase (18)(19)(20), and recently, the B family pol gp43 from bacteriophage RB69 (21). All share a functional polymerase structure, which resembles a right hand built by the palm, fingers and thumb domains (see ref. 7 for review). Although the fingers and thumb domains are highly diverse among the different families, the palm domains, which contain the conserved catalytic aspartate residues, show a similar topology among all families except pol ␤. The polymerase nucleotidyl transfer was studied in detail for the A family polymerases, HIV reverse transcriptase, and pol ␤, and was shown to involve two metal ions (summarized in ref. 7).Considerably less is known for the family of type B pols, which are replicative enzymes in eukaryotes and most likely also Archaea (22,23). The structure of gp43 from bacteriophage RB69 (21) provided an excellent first insight into this family. In addition to the three polymerase domains, gp43 contains an 3Ј 3 5Ј exonuclease domain and an N-termin...

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The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA.

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