Resolving the Kinetic States of a Proofreading DNA Polymerase

Abstract

DNA polymerase (DNAp) carries out DNA replication. To ensure high fidelity in replication it is selective in incorporating correct nucleotides and in correcting mismatches. Using single molecule tools we have investigated the kinetic states of DNAp molecules and have proposed a kinetic model describing the interplay between replication and proofreading of DNA polymerase [Tjalle P. Hoekstra, 2014]. Based on new data we have refined the previous kinetics models of T7 DNAp and have found evidence for the existence of previously unidentified pausing states.The model predicts that the short pauses are related to DNAp concentration and that exonucleolysis activity of DNAp is force independent. To test the validity of our model, we investigated the impact of low DNAp concentration (less than 10 nM) and low force (less than 6 pN).Our new results indicate as predicted that the duration of short pauses increases when DNAp concentration is reduced. Interestingly we also find the existence of a previously unobserved very long paused state. The nature of this pause state still needs to be determined. We characterized the behavior of DNAp at low force in a parallelized manner using a newly developed Acoustic Force Spectroscopy (AFS) method. These experiments reveal that exonucleolysis events indeed occur at low force as predicted by our model. The confirmation of both predictions validates our model and sheds further insight into the detailed kinetics of DNAp.