Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius

Abstract

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...

Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

“…The comparable residues in class B polymerases are located in the fingers subdomain and may face toward the polymerase cleft (23)(24)(25)(26). Mutations of the essential Lys greatly reduce polymerase activity in human pol ␣, 29 pol,, and Klenow fragment (5, 6).…”

Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

“…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).…”

“…1). Furthermore, steadystate kinetic studies have identified hyperthermophilic DNA polymerase residues important for polymerization and exonuclease activities and for nucleotide binding (18,29,(31)(32)(33)(34)(35). Nucleotide analogs have also been important in identifying dNTP recognition determinants important in the polymerase reaction (32-36) and have proven useful in a variety of molecular biology applications, such as DNA sequencing and detection of single nucleotide polymorphisms (37-41).…”

“…Family B DNA polymerases from hyperthermophilic Archaea Thermococcus sp. 9°N (22), Thermococcus gorgonarius (18), and Pyrococcus kodakaraensis KOD1 (24) and mesophilic bacteriophage RB69 (23) have high sequence and structural homologies and provide a framework for analysis of active site structure and function in this enzyme family (Fig. 1).…”

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

“…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).…”