Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 ''steric gate'' can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.

To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3-5-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzymesubstrate interactions and is not easily perturbed. In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site. Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, 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 mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions. The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T⅐G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A.

We have employed site-directed mutagenesis to identify those amino acid residues that interact with the deoxynucleoside triphosphate (dNTP) and pyrophosphate in the Klenow fragment-DNA-dNTP ternary complex. Earlier structural, mutagenesis, and labeling studies have suggested that the incoming dNTP molecule contacts a region on one side of the polymerase cleft, primarily involving residues within the so-called "fingers" subdomain. We have made mutations in residues seen to be close to the dNTP in the crystal structure of the Klenow fragment-dNTP binary complex and have examined their kinetic parameters, particularly Km(dNTP). The results are consistent with the notion that there are significant differences between the dNTP interactions in the binary and ternary complexes, although some contacts may be present in both. When dTTP is the incoming nucleotide, the side chains of Arg754 and Phe762 make the largest contributions to binding; measurement of Km(PPi) suggests that Arg754 contacts the beta- or gamma-phosphate of the dNTP. With dGTP, the contribution of Arg754 remains the same, but the additional interactions are provided by both Lys758 and Phe762, suggesting that the binding of the incoming dNTP is not identical under all circumstances. Mutations in Arg754 and Lys758 also cause a substantial decrease in the rate of polymerase-catalyzed incorporation, and sulfur elemental effect measurements indicate that loss of Arg754 (and perhaps also Lys758) slows the rate of the chemical step of the reaction. Mutations of Arg682, His734, and Tyr766 affect the binding of DNA, suggesting that these mutations, whose effect on dNTP binding is small, may influence dNTP binding indirectly via the positioning of the DNA template-primer.

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In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two ␣ helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590 -613) that removes a portion of the tip of the thumb. The mutant polymerase has relatively normal dNTP binding and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced. Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by templateprimer strand slippage. These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.The Klenow fragment, 1 a 68-kDa carboxyl-terminal fragment of Escherichia coli DNA polymerase I, contains both DNA polymerase and 3Ј 3 5Ј exonuclease activity. The polymerase active site is on a 45-kDa COOH-terminal domain of Klenow fragment, and the 3Ј 3 5Ј proofreading exonuclease active site is on the smaller NH 2 -terminal domain. Structural information obtained from crystal structures of Klenow fragment demonstrates that the polymerase domain resembles a partially open right hand with three subdomains that form a cleft (for review, see Joyce and Steitz (1994)). The palm is located at the base of the cleft and contains the catalytically important carboxylates. The fingers form one wall of the cleft and the thumb another.