Single-molecule FRET reveals proofreading complexes in the large fragment of <em>Bacillus stearothermophilus</em> DNA polymerase I

Abstract

There is increasing interest in the use of DNA polymerases (DNA pols) in next-generation sequencing strategies. These methodologies typically rely on members of the A and B family of DNA polymerases that are classified as high-fidelity DNA polymerases. These enzymes possess the ability to selectively incorporate the correct nucleotide opposite a templating base with an error frequency of only 1 in 106 insertion events. How they achieve this remarkable fidelity has been the subject of numerous investigations, yet the mechanism by which these enzymes achieve this level of accuracy remains elusive. Several smFRET assays were designed to monitor the conformational changes associated with the nucleotide selection mechanism(s) employed by DNA pols. smFRET has also been used to monitor the movement of DNA pols along a DNA substrate as well as to observe the formation of proof-reading complexes. One member among this class of enzymes, the large fragment of Bacillus stearothermophilus DNA polymerase I (Bst pol I LF), contains both 5′→3′ polymerase and 3′→5′ exonuclease domains, but reportedly lacks exonuclease activity. We have designed a smFRET assay showing that Bst pol I LF forms proofreading complexes. The formation of proofreading complexes at the single molecule level is strongly influenced by the presence of the 3′ hydroxyl at the primer-terminus of the DNA substrate. Our assays also identify an additional state, observed in the presence of a mismatched primer-template terminus, that may be involved in the transfer of the primer-terminus from the polymerase to the exonuclease active site.

Deep Dive Into the DNA Polymerase Through Insilico Analysis: An Information to Get Better PCR Enzyme From the Ancient One

“…Researchers assessed the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (formerly Pyrococcus kodakaraensis KOD1)'s 3'-5'exonuclease (proofreading) activity and PCR performance (Kuroita et al, 2005). Christian et al (2018) observed the assembly of proofreading complexes and the migration of DNA pols of Geobacillus stearothermophilus along a DNA substrate by using smFRET.…”