Does T7 DNA Polymerase Backtrack During Proofreading?

Abstract

DNA replication is an essential cell process in which the genetic information is copied by replicative DNA polymerases (DNAp). The molecular basis of DNA replication is the addition of nucleotides by DNAp to a growing primer, using single-stranded DNA as a template. High fidelity of the processive T7 DNA polymerase comes from nucleotide selection at the polymerase active site, but is increased several orders of magnitude by an additional intrinsic proofreading ability. In this kinetic process, a partly melted primer shuttles to the exonuclease active site where incorporated mismatches are excised. After excision of erroneous nucleotides, the trimmed primer can shuttle back to the polymerase active site to resume replication. Elucidating the mechanism of the shuttling between these two activities of DNAp is essential for understanding the proofreading mechanism of DNA polymerases.Transfer of the primer to the exonuclease active site is induced by disruption of the primer-template structure upon the incorporation of a mismatch. Application of tension to the DNA also destabilizes the primer-template structure and can therefore be used to shift the fine-tuned balance between polymerization and proofreading (Wuite et al, 2001; Ibarra et al, 2009).Using optical tweezers, we study the kinetic coordination between exonuclease and polymerase activities, while applying different tensions. In these experiments we observe an additional waiting state between proofreading activities, during which the DNAp remains bound to the DNA. The force-dependent rate out of this state suggests that DNAp enters a state comparable to RNA polymerase backtracked state, which was shown to play a role in tuning the fidelity (Shaevitz et al, 2003). We speculate that our observed waiting state might play a similar role in the fidelity of DNA polymerase.