Abstract 1968 Dynamics of Exonuclease Proofreading by T7 DNA Polymerase During DNA Replication

In this study we explore the interplay of polymerase and exonuclease active sites of T7 DNA polymerase in selective error correction during high fidelity DNA replication. First, we characterize the substrate specificity of the proofreading exonuclease domain in a high-fidelity DNA polymerase, showing efficient proofreading of terminal mismatches and surprisingly, even greater efficiency for mismatches buried by correct bases suggesting that the polymerase may correct some mistakes by first extending them with the correct base. We then developed a homology model for the DNA primer strand in the exonuclease active site showing that the DNA must backtrack and 3 bases melt for the primer to partition to the exonuclease active site for hydrolysis. Based on these results we designed a DNA substrate containing the fluorescent cytosine analog tCo at the n-2 position to measure kinetics of proofreading on physiologically relevant substrates. Combining pre-steady state stopped flow fluorescence measurements of strand separation with single turnover rapid quench experiments allowed us to obtain a global fit to elucidate the rate constants and kinetic mechanism for high fidelity error correction. These studies revealed a unique intermediate state and stimulation of transfer to the exo site by nucleoside triphosphates. Hydrolysis-resistant oligonucleotides track DNA transfer without hydrolysis. Our proposed model explains faster removal of buried mismatches than single 3′-terminal mismatches, providing an additional avenue for error correction. This comprehensive model demonstrates how DNA transfer, coupled with base excision, facilitates efficient selective mismatch removal during DNA replication, enhancing fidelity by over 1000-fold.