Publication: Probing the conformational landscape of DNA polymerases using diffusion-based single-molecule FRET

J. Hohlbein and A.N. Kapanidis, Methods in Enzymology: Single-molecule Enzymology Part A & B, 581, 353-378, 2016, [link]

Monitoring conformationalchanges in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and the closed conformations of DNA polymerases, our smFRET assays utilising doubly labelled variants of E. coli DNA polymerase I were pivotal in identifying and characterising a partially-closed conformation as a primary checkpoint for nucleotide selection. Here, we provide a comprehensive overview of the methods we used for the conformational analysis of wild-type DNA polymerase and some of its low-fidelity derivatives; these methods include strategies for protein labelling and our procedures for solution-based single-molecule fluorescence data acquisition and data analysis. We also discuss alternative single-molecule fluorescence strategies for analysing the conformations of DNA polymerases in vitro and in vivo.