PMID:11602576
| Citation |
Shinkai, A and Loeb, LA (2001) In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection. J. Biol. Chem. 276:46759-64 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M104780200 |
| Keywords |
Amino Acid Motifs; Base Pair Mismatch; Binding Sites; Codon, Terminator; DNA Polymerase I/metabolism; Escherichia coli/enzymology; Genes, Reporter; Kinetics; Mutagenesis, Site-Directed; Mutation; Plasmids/metabolism; Tryptophan/chemistry; beta-Lactamases/metabolism |
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