A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity

J Biol Chem. 2001 Feb 16;276(7):5044-51. doi: 10.1074/jbc.M008701200. Epub 2000 Nov 7.


DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Base Pair Mismatch
  • Binding Sites
  • DNA Replication*
  • Evolution, Molecular
  • Genes, Bacterial
  • Isoleucine / genetics
  • Kinetics
  • Models, Molecular
  • Mutation
  • Polymerase Chain Reaction
  • Structure-Activity Relationship
  • Taq Polymerase / chemistry
  • Taq Polymerase / genetics*
  • Taq Polymerase / metabolism*
  • Templates, Genetic


  • Isoleucine
  • Taq Polymerase