Crystal Structure of a Thermostable Type B DNA Polymerase From Thermococcus Gorgonarius

Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3600-5. doi: 10.1073/pnas.96.7.3600.

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 A 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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cloning, Molecular
  • Computer Graphics
  • Conserved Sequence
  • Crystallography, X-Ray / methods
  • DNA Polymerase I / chemistry*
  • DNA Polymerase I / metabolism
  • Enzyme Stability
  • Hot Temperature
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Secondary*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Thermococcus / enzymology*
  • Thermodynamics

Substances

  • Recombinant Proteins
  • DNA Polymerase I

Associated data

  • PDB/1TGO