DEOXYNUCLEOSIDE TRIPHOSPHATE AND PYROPHOSPHATE BINDING-SITES IN THE CATALYTICALLY COMPETENT TERNARY COMPLEX FOR THE POLYMERASE REACTION CATALYZED BY DNA-POLYMERASE-I (KLENOW FRAGMENT)

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 K-m(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 Arg(754) and phe(762) make the largest contributions to binding; measurement of K-m(PPi) suggests that Arg(754) contacts the beta- or gamma-phosphate of the dNTP. With dGTP, the contribution of Arg(754) remains the same, but the additional interactions are provided by both Lys(758) and phe(762), suggesting that the binding of the incoming dNTP is not identical under all circumstances. Mutations in Arg(754) and Lys(758) also cause a substantial decrease in the rate of polymerase-catalyzed incorporation, and sulfur elemental effect measurements indicate that loss of Arg(754) (and perhaps also Lys(758)) slows the rate of the chemical step of the reaction, Mutations of Arg(682), His(734) and Tyr(766) 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.