In Vivo Mutagenesis by Escherichia coliDNA Polymerase I

Abstract

The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColEl-type origin and an ochre codon in the β-lactamase gene. We screened 53 single mutants within the region Val700-Arg712 in the polymerase active-site motif A. Only replacement of Ile709 yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the β-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent Km values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3′-5′ exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3′-5′ exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.