Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment)

To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in t he polymerase domain of a 3'-5'-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity simi lar to that of the wild-type control, suggesting that fidelity at the polym erase active site depends on highly specific enzyme-substrate interactions and is not easily perturbed. In addition to the previously studied Y766A mu tator, four novel base substitution mutators were identified; they are R668 A R682A, E710A, and N845A, Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exp osed surface of the polymerase cleft near the polymerase active site. Analy sis of base substitution errors at four template positions indicated that e ach of the five mutator polymerases has its own characteristic error specif icity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. We separated the contributions of the nucleotide insertion and misma tch extension steps by using a novel fidelity assay that scores base substi tution errors during synthesis to fill a single nucleotide gap land hence d oes not require mismatch extension) and by measuring the rates of polymeras e-catalyzed mismatch extension reactions. The R682A, E710A, Y766A and N845A mutations cause decreased fidelity at the nucleotide insertion step, where as R668A results in lower fidelity in both nucleotide insertion and mismatc h extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a TG mismatch under the conditions of the fidelity assay, providing one explanation for the ant i-mutator phenotypes of mutants such as R754A and Q849A.