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 aci d residues that interact with the deoxynucleoside triphosphate (dNTP) and pyrophosphate in the Klenow fragment-DNA-dNTP ternary complex, Ear lier structural, mutagenesis, and labeling studies have suggested that the incoming dNTP molecule contacts a region on one side of the polym erase cleft, primarily involving residues within the so-called ''finge rs'' 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(d NTP). The results are consistent with the notion that there are signif icant differences between the dNTP interactions in the binary and tern ary complexes, although some contacts may be present in both, When dTT P is the incoming nucleotide, the side chains of Arg(754) and phe(762) make the largest contributions to binding; measurement of K-m(PPi) su ggests that Arg(754) contacts the beta- or gamma-phosphate of the dNTP . With dGTP, the contribution of Arg(754) remains the same, but the ad ditional interactions are provided by both Lys(758) and phe(762), sugg esting that the binding of the incoming dNTP is not identical under al l circumstances. Mutations in Arg(754) and Lys(758) also cause a subst antial 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 bind ing of DNA, suggesting that these mutations, whose effect on dNTP bind ing is small, may influence dNTP binding indirectly via the positionin g of the DNA template-primer.