title: A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity.
abstract: 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.
authors.affiliation: Joseph Gottstein Memorial Cancer Laboratory, Departments of Pathology and Biological Structure, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: P H
authors.initials: PH
authors.lastname: Patel
authors.forename: H
authors.initials: H
authors.lastname: Kawate
authors.forename: E
authors.initials: E
authors.lastname: Adman
authors.forename: M
authors.initials: M
authors.lastname: Ashbach
authors.forename: L A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D020816:Amino Acid Motifs; D020137:Base Pair Mismatch; D001665:Binding Sites; D004261:DNA Replication; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D007532:Isoleucine; D019914:Taq Polymerase