We present a kinetic partitioning mechanism by which the highly efficient 3'-->5' exonuclease activity of T7 DNA polymerase maximizes its contribution to replication fidelity with minimal excision of correctly base-paired DNA. The elementary rate constants for the proposed mechanism have been measured directly from single-turnover experiments by using rapid chemical quench-flow techniques. The exonuclease activity of T7 DNA polymerase toward single-stranded DNA is quite fast (k(x) > 700 s-1). This rapid exonuclease is restrained with double-stranded DNA by a kinetic partitioning mechanism that favors the binding of the DNA to the polymerase site to prevent the rapid degradation of matched DNA and yet allows selective removal of mismatched DNAs. Both matched and mismatched DNAs bind tightly to the polymerase site, with approximately equal affinities, K(d)p = 20 and 10 nM, respectively. Selective removal of the mismatch is governed by the rate of transfer of the DNA from the polymerase to the exonuclease site (k(p)-->x). The rapid excision of matched DNA is limited by a slow transfer rate (k(p)-->x = 0.2 s-1) from the polymerase to the exonuclease site relative to the rate of polymerization [k(p) = 300 s-1; Patel et al. (1991) Biochemistry (first of three papers in this issue)]. Removal of mismatched DNA is facilitated by its faster transfer rate (k(p)-->x = 2.3 s-1) to the exonuclease site relative to the slow rate of polymerization over a mismatch [k(p)i = 0.012 s-1; Wong et al. (1991) Biochemistry (second of three papers in this issue)]. The dissociation rate of the mismatched DNA is slower (k(off) = 0.4 s-1) than the observed transfer rate (2.3 s-1); thus, an intramolecular transfer of mismatched DNA from the polymerase to the exonuclease site was observed. Bidirectional transfer was observed as the matched DNA generated by exonucleolytic hydrolysis of the mismatched primer/terminus was rapidly transferred from the exonuclease site to the polymerase site (k(x)-->p > 700 s-1). Approximately 50% of the mismatched DNA that dissociates rather than transferring intramolecularly to the exonuclease site is also rapidly excised because it binds more rapidly to the exonuclease site than to the polymerase site (k(on)x = 5 X 10(8) M-1 s-1; k(on)p = 4 X 10(7) M-1 s-1). The slow polymerization rate onto a mismatch provides time during which an error can be corrected by the kinetic partitioning mechanism. The contribution of the exonuclease activity to fidelity, represented by phi-x, can be calculated as [GRAPHICS] where k(off) is the dissociation rate of the DNA from the polymerase site into free solution, k(p)-->x is the transfer rate of the DNA from the polymerase site to the exonuclease site Kp is the polymerization rate onto a correctly base-paired DNA, k(p)i is the polymerization rate onto a mismatch, and theta is the fraction of DNA excised rapidly upon rebinding. Under our conditions, the cost of this proofreading is minimal, with the loss of only a small fraction of correctly base-paired products, 0.0008.
