# Angstrom-resolution single-molecule fluorescence resonance energy transfer reveals mechanisms of DNA helicases

## TL;DR

This study enhances single-molecule FRET resolution to 0.5 base pairs using a nanotensioner, enabling detailed analysis of DNA unwinding mechanisms of different helicases at the single-base level.

## Contribution

It introduces a nanotensioner method that significantly improves FRET resolution, revealing distinct unwinding stepping behaviors of yeast Pif1 and E. coli RecQ helicases.

## Key findings

- Pif1 exhibits 1-bp stepping kinetics.
- RecQ breaks 1 bp at a time but releases nucleotides randomly.
- High-resolution data supports a new quantitative unwinding model.

## Abstract

Single-molecule FRET is widely used to study helicases by detecting distance changes between a fluorescent donor and an acceptor anchored to overhangs of a forked DNA duplex. However, it has lacked single-base pair (1-bp) resolution required for revealing stepping dynamics in unwinding because FRET signals are usually blurred by thermal fluctuations of the overhangs. We designed a nanotensioner in which a short DNA is bent to exert a force on the overhangs, just as in optical/magnetic tweezers. The strategy improved the resolution of FRET to 0.5 bp, high enough to uncover the differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but questers the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.