Proofreading of mismatches within primer-template junctions by Escherichia coli DNA polymerase I in vitro and in vivo

Abstract

Escherichia coli DNA polymerase I (Pol I) possesses a 3’ to 5’ proofreading function. Using a non-inhibitory in vitro proofreading assay and MALDI-TOF MS analysis, we demonstrated the Pol I proofreading function was effective at removal of mismatches within the primer-template junction. Mismatches of 1–4 nucleotides (nt) from the primer 3′ end could be completely or partially corrected, with no additional editing observed further upstream. A backward movement mechanism was proposed involving distributive backtracking of polymerase along the template to remove non-fully complemented primers in order for DNA synthesis to recover. Co-editing DNA substrates containing two mismatches, one at 1–4-nt of the primer 3’ end and the other outside of normal proofreading range, confirmed our distributive backtracking hypothesis. Additionally, a time course analysis revealed proofreading of internal mismatches was a non-processive reaction. To further confirm the validity of our proofreading model, we used in vivo, phagemid-derived nicked C-C substrates. Transformation results were consistent with the notion that mismatches located less than 4-nt upstream of the 3′ end could be successfully proofread. In vivo proofreading of double mismatches also supports our model of polymerase backtracking for internal mismatch editing.