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

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 ␤-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.DNA polymerases function in DNA replication, repair, and recombination and are essential for maintaining the integrity of the genome. Multiple DNA polymerases have been found in prokaryotes, eukaryotes, and viruses and may have different properties, including variations in the accuracy, or fidelity, of DNA synthesis (1-5). Polymerases that replicate large genomes typically have high fidelity, with error rates on the order of 10 Ϫ5to 10 Ϫ6 (6 -8) to prevent the accumulation of deleterious mutations in the genome. Deficits in DNA polymerase fidelity have been associated with cancer, accelerated aging, and infertility (9 -11).Mutator and antimutator DNA polymerases harbor amino acid substitutions resulting in decreased and increased fidelity, respectively. These variant enzymes provide insights into the structural basis of accurate DNA synthesis (12-18). Many mutators have been identified due to the availability of powerful selection methods (19). The majority of studied mutators harbor amino acid substitutions in well conserved polymerase motifs. Mutator polymerases have also been used extensively in biotechnological applications, such as DNA sequencing and error-prone PCR (20). Far fewer antimutator enzymes have been identified due to the lack of selection methods. The ones identified thus far either exh...

A Substitution in the Fingers Domain of DNA Polymerase δ Reduces Fidelity by Altering Nucleotide Discrimination in the Catalytic Site*

“…At the Arg 696 site alone, we identified 13 amino acids representing polar, nonpolar, charged, and hydrophobic residues that could substitute for alanine. This is in contrast to previous work on Escherichia coli Pol I and TaqDNA polymerase using random mutagenesis of the entire polymerase molecule which suggested that this position is very highly conserved (28,29). The previously described mutation, R689W, which was identified in the human colorectal cancer cell line, DLD-1, has been demonstrated to be a strong mutator that is non-viable in haploid yeast (23).…”

Genetic Constraints on Protein Evolution

“…The presence of one mutation was found to significantly increase the chances of another mutation at a structurally-interacting site. In the case of ionic interactions, the increase was almost 4-fold (Loh et al, 2007b). This is strong evidence of a role of compensatory mutations in shaping protein evolution.…”

“…Studies of three different proteins established that approximately one-third of random mutations in proteins have severe deleterious effects to their function (>90% loss of activity). Two of these are monomers, human 3-methyladenine DNA glycosylase (AAG, 298 amino acids long) (Guo et al, 2004a), and the 430 amino acids of the E. coli Pol I Kleenow fragment (Loh et al, 2007b). The third one is the E. coli lac repressor, a tetramer of four 360 amino acid polypeptides.…”

“…Several studies have shown that principles of protein folding and solubility constrain protein evolution in similar manners. In general, residues located on the surface of globular proteins are more tolerant of amino acid substitutions (Loh et al, 2007b;Pagel et al, 2006;Suckow et al, 1996). The hydrophobic protein core tends to be less tolerant to mutations due to the need for stable packing of atoms, to the limited availability of stabilizing bonds, and to the increased likelihood that these residues are involved in catalysis.…”

Optimization of DNA polymerase mutation rates during bacterial evolution

“…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.…”