title: Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.
abstract: We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.
authors.affiliation: The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, WA 98195-7705, USA.
authors.forename: Manel
authors.initials: M
authors.lastname: Camps
authors.forename: Jussi
authors.initials: J
authors.lastname: Naukkarinen
authors.forename: Ben P
authors.initials: BP
authors.lastname: Johnson
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001398:Aztreonam / Q000494:pharmacology; D001483:Base Sequence; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005818:Genetic Engineering; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004256:DNA Polymerase I; D001618:beta-Lactamases; D001398:Aztreonam