In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.
The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) 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.
Shinkai
A
A
Department of Pathology, Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, WA 98195-7705, USA.
Loeb
L A
LA
D020816:Amino Acid Motifs; D020137:Base Pair Mismatch; D001665:Binding Sites; D018388:Codon, Terminator; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D017930:Genes, Reporter; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D001618:beta-Lactamases / Q000378:metabolism
D018388:Codon, Terminator; D014364:Tryptophan; D004256:DNA Polymerase I; D001618:beta-Lactamases