Highly Tolerated Amino Acid Substitutions Increase the Fidelity of Escherichia coli DNA Polymerase I

Ern Loh,Juno Choe,Lawrence A Loeb

doi:10.1074/jbc.m611294200

Abstract

Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy (antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a beta-lactamase reporter. Mutation frequencies varied from 1/1000- to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.

Highlights

To 10Ϫ6 (6 – 8) to prevent the accumulation of deleterious mutations in the genome
This was attained by conducting a comprehensive analysis of amino acid substitutions in polymerase I (Pol I) that result in antimutator or mutator activity
Because we wanted to focus on mutations in the polymerase domain, nucleotides at positions 1493–2784 that encode the polymerase catalytic domain were randomly substituted, and the 3Ј-5Ј exonuclease proofreading activity was inactivated by a D424A substitution [27, 37]

Summary

EXPERIMENTAL PROCEDURES

Escherichia coli strain JS200 (SC-18 recA718 polA12 uvrA155 trpE6 lon-11 sulA1) was first described as SC18-12. Assay for Pol I Polymerase Activity—JS200 cells containing pECpolI mutants were grown in LB at 30 °C with 1 mM isopropyl 1-thio-␤-D-galactopyranoside to induce protein expression. Protein Purification—Mutant and wild-type Klenow fragments, encompassing the 3Ј–5Ј exonuclease domain and the polymerase domain, were subcloned into the pLEX vector (Invitrogen), expressed, and purified as previously described [27]. This Pol I construct included a six-histidine N terminus tag. M13 Gapped Assay—We characterized the DNA synthesis fidelity of purified mutant and wild-type polymerase in vitro on the M13 gapped forward mutation assay, as previously described [36]. The complete fill-in of the M13 gapped substrate was monitored by gel (data not shown) and by the consistency of mutation rates after incubation with increased amounts of the DNA polymerase

RESULTS

Mean substitutability indices of Pol I regions

Wild type