Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity

Genet Anal. 1996 Mar;12(5-6):185-95.


A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA Polymerase I / genetics
  • DNA Polymerase I / isolation & purification
  • DNA Polymerase I / metabolism*
  • DNA Primers / genetics
  • DNA Repair
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Enzyme Stability
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / isolation & purification
  • Exodeoxyribonucleases / metabolism
  • Genes, Bacterial
  • Geobacillus stearothermophilus / enzymology
  • Geobacillus stearothermophilus / genetics
  • Molecular Sequence Data
  • Molecular Weight
  • Sequence Homology, Amino Acid
  • Temperature


  • DNA Primers
  • DNA, Bacterial
  • DNA Polymerase I
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V

Associated data

  • GENBANK/U33536