Mechanism of proofreading and strand displacement of a family B DNA Polymerase (φ29 DNA Polymerase) by single-molecule FRET

Abstract

High-fidelity DNA polymerases (DNAPs) precisely coordinate the activities of polymerase (pol) and exonuclease (exo), but the rules governing conformational control remains unclear. In the first part of this thesis, we examined the full course of proofreading activity by single molecule fluorescence imaging. We found that, in contrast to the current textbook model, DNAP continues to polymerize a few additional nucleotides without correcting the error immediately after incorporating an incorrect nucleotide, introducing a mismatch. Consequently, as soon as the mismatched base reaches the third position behind the primertemplate (pt)-junction, all the pt-junctions immediately melt, which is called a DNA melting singularity. The singularity is the point at which the melting energy is minimum at the pt-junction with a single mismatch, which suddenly triggers the conformational transition from pol to exo. Our study provides a novel mechanism by which DNAPs convert their mistakes to thermodynamic advantages to achieve high fidelity DNA replication. In the second part of this thesis, we focused on study the strand displacement activity. We found that (1) there is the movement of TPR2 domain forming the 2 confirmations in the φ29 DNAP for the pol and strand displacement activities, (2) the conformational change triggers and stabilizes by the displaced DNA created during the strand displacement, (3)the polymerase encounter the duplex DNA (“initiation”), the unwinding of duplex assists by the aromatic ring residues (Y405, F414), and hydrophobic residues (L406, L412) followed by “the processive” strand displacement.