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 ؋ 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 ␤-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 ␤-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. DNA polymerases catalyze the template-directed incorporation of deoxynucleotides into a growing primer terminus. Errors in nucleotide incorporation by DNA polymerases are significant sources of mutation, and contribute to genetic diversity. The fidelity of replication by DNA polymerases is ensured by base pair complementarity, by dNTP-induced conformational changes, and, in some cases, by proofreading catalyzed by a 3Ј 3 5Ј exonuclease domain (1).DNA polymerase I (Pol I) is one of five different DNA polymerases that have been described so far in Escherichia coli. In vivo, Pol I plays a role in lagging-strand replication of chromosomal DNA, DNA repair, and ColE1 plasmid replication (2). Pol I has been intensively studied as a model polymerase, and extrapolation to a diversity of other DNA polymerases has been possible given the conservation in structure and catalytic mechanism across eukaryotic and prokaryotic DNA polymerases (3).Here, we engineered a highly error-prone E. coli DNA Pol I, and used this mutant polymerase as the basis of an in vivo mutagenesis system for modification of targeted sequences. Generation of enzyme variants is important in both basic studies of enzyme structure and function, and in creation of improved derivatives for medicine and technology. Several approaches for enzyme modification have been described to date. One of these approaches is random nucleotide mutagenesis, which involves the generation of large libraries harboring random mutations in vitro (4) and the subsequent identification of mutants of inter...

Optimization of DNA polymerase mutation rates during bacterial evolution

“…This result was unanticipated. We had expected their high mutation rate to provide a selective advantage, as had previously been demonstrated in antibiotic-facilitated bottlenecks with this degree of mutator (25). These results demonstrate a limit to the selective advantage that can be conferred by enhanced mutagenesis.…”

“…Mutants displaying altered replication fidelities were initially identified in an in vivo screen on the basis of the reversion of a plasmid-borne β-lactamase gene. The effect of PolI mutants on elevating chromosomal mutagenesis was confirmed by using reversion of a chromosomally encoded trpE65 (ochre) allele (24) and/or a forward mutation assay for rifampicin resistance (25). The per-base-pair rate of chromosomal mutagenesis by individual PolI mutator polymerases is approximately 10-fold lower than that obtained using reporter plasmid DNA (24,25).…”

“…The effect of PolI mutants on elevating chromosomal mutagenesis was confirmed by using reversion of a chromosomally encoded trpE65 (ochre) allele (24) and/or a forward mutation assay for rifampicin resistance (25). The per-base-pair rate of chromosomal mutagenesis by individual PolI mutator polymerases is approximately 10-fold lower than that obtained using reporter plasmid DNA (24,25). This is presumably due to the fact that only a portion of the E. coli genome is replicated by PolI.…”

“…We have created a large collection of PolI mutants that exhibit either an elevated or a reduced in vivo mutation rate, with a representation of fidelities spanning six orders of magnitude (23)(24)(25)(26). Here we report on the use of these mutants in competition experiments to investigate the relationship between replication fidelity and evolutionary survival.…”

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

“…Major advantages of the viral evolution approach are the continuous cascade of replication cycles and the continuous generation of genetic diversity. Camps et al (33) recently presented a targeted gene evolution approach in E. coli with an error-prone DNA polymerase I. These bacteria were used to modify an antibiotic resistance gene and to select for improved variants.…”