title: DNA polymerase active site is highly mutable: evolutionary consequences.
abstract: DNA polymerases contain active sites that are structurally superimposable and highly conserved in sequence. To assess the significance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of Thermus aquaticus DNA polymerase I. After selection, by using genetic complementation, we obtained a library of approximately 8, 000 active mutant DNA polymerases, of which 350 were sequenced and analyzed. This is the largest collection of physiologically active polymerase mutants. We find that all residues of motif A, except one (Asp-610), are mutable while preserving wild-type activity. A wide variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative substitutions predominate at regions that stabilize tertiary structures. Several mutants exhibit unique properties, including DNA polymerase activity higher than the wild-type enzyme or the ability to incorporate ribonucleotide analogs. Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. The high mutability of the polymerase active site in vivo and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis. The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.
authors.affiliation: The Joseph Gottstein Memorial Cancer Laboratory, Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195-7705, USA.
authors.forename: P H
authors.initials: PH
authors.lastname: Patel
authors.forename: L A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013824:Thermus / Q000201:enzymology
chemical_list: D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase