Conformational Dynamics of DNA Polymerases Revealed at the Single-Molecule Level.

David P Millar

doi:10.3389/fmolb.2022.826593

Abstract

DNA polymerases are intrinsically dynamic macromolecular machines. The purpose of this review is to describe the single-molecule Förster resonance energy transfer (smFRET) methods that are used to probe the conformational dynamics of DNA polymerases, focusing on E. coli DNA polymerase I. The studies reviewed here reveal the conformational dynamics underpinning the nucleotide selection, proofreading and 5′ nuclease activities of Pol I. Moreover, the mechanisms revealed for Pol I are likely employed across the DNA polymerase family. smFRET methods have also been used to examine other aspects of DNA polymerase activity.

Highlights

DNA polymerases are intrinsically dynamic macromolecular machines
The purpose of this review is to describe the single-molecule Förster resonance energy transfer (smFRET) methods that are used to probe the conformational dynamics of DNA polymerases
The results reveal the different physical mechanisms employed in each case: 1) nucleotide selection is coupled to movement of the fingers domain, 2) proofreading involves physical movement of the DNA substrate between separated pol and exo sites, and 3) the 5′ nuc activity requires both movement of DNA and a large conformational change of the polymerase

Summary

Introduction

During elongation of a nascent DNA strand, a polymerase must select the correct nucleotide substrate, adopt a conformation that promotes catalysis of the phosphoryl transfer reaction, release the pyrophosphate by product and move to the templating position. This sequence of events likely involves dynamic conformational changes of the polymerase-DNA complex. While structural studies have provided high resolution snapshots of DNA polymerases in specific functional states (Doublié et al, 1998; Kiefer et al, 1998; Johnson and Beese, 2004; Zahn et al, 2015; Huang et al, 2018; Jain et al, 2018; Jain et al, 2019; Hoitsma et al, 2020), much less is known about the conformational dynamics that underpin DNA polymerase activity

Objectives

Conclusion