| DNA polymerases catalyze the synthesis of DNA and function in the replication, repair, and recombination of the genome. The work presented in this thesis delves into two aspects of DNA polymerases: (1) structural substitutions that create highly active antimutators and (2) the optimization of polymerase fidelity during evolution.; Many antimutators have been observed but these mainly involve increased exonuclease proofreading or dramatically decreased polymerase activity. To identify antimutators that retain high polymerase activity fidelity, we created a DNA polymerase I (Pol I) mutant library, with random substitutions across the entire polymerase domain. This library was pre-selected for activity and screened for significant alterations in replication accuracy. We report that the polymerase domain tolerates two-thirds of mutations, and plasticity often occurs at evolutionarily conserved regions. Antimutator polymerases constituted 12% of the variant polymerase population, and many of these displayed high polymerase activity. 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 position 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.; Mutation rates in nature typically occur in a narrow range. To determine whether it is experimentally possible to observe the selection of an optimum mutation rate, we generated a panel of 66 DNA Pol I mutants in E. coli with a wide range of DNA replication fidelities spanning 3 orders of magnitude higher and lower than wild-type and competed them for 350 generations in six independently passaged cultures. Subsequent sequencing of 96 isolates revealed that although the identity of the dominant mutant in each culture varied, survival was nevertheless correlated with a narrow range of fidelities, 8- to 33-fold above wild-type. Mutants exhibiting mutation rates 120- and 900-fold above wild-type were not detected in the surviving population, nor were wild-type or antimutator polymerases. The growth phenotype of the dominant mutant after competition varied between cultures, which is consistent with the "hitchhiking" model of how mutator alleles confer selective advantage. |
