Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication

We show that T7 DNA polymerase (pol) and exonuclease (exo) domains contribute to selective error correction during DNA replication by regulating bidirectional strand transfer between the two active sites. To explore the kinetic basis for selective removal of mismatches, we used a fluorescent cytosine analog (1,3-diaza-2-oxophenoxazine) to monitor the kinetics of DNA transfer between the exo and pol sites. We globally fit stopped-flow fluorescence and base excision kinetic data and compared results obtained with ssDNA versus duplex DNA to resolve how DNA transfer governs exo specificity. We performed parallel studies using hydrolysis-resistant phosphorothioate oligonucleotides to monitor DNA transfer to the exo site without hydrolysis. ssDNA binds to the exo site at the diffusion limit (10(9) M(−1) s(−1), K(d) = 40 nM) followed by fast hydrolysis of the 3′-terminal nucleotide (>5000 s(−1)). Analysis using duplex DNA with a 3′-terminal mismatch or a buried mismatch exposed a unique intermediate state between pol and exo active sites and revealed that transfer via the intermediate to the exo site is stimulated by free nucleoside triphosphates. Transfer from the exo site back to the pol site after cleavage is fast and efficient. We propose a model to explain why buried mismatches are removed faster than single 3′-terminal mismatches and thereby provide an additional opportunity for error correction. Our data provide the first comprehensive model to explain how DNA transfer from pol to exo active sites and back again after base excision allow efficient selective mismatch removal during DNA replication to improve fidelity by more than 1000-fold.