High-Resolution Single-Molecule Analysis of UvrD Helicase using Nanopore Tweezers

Abstract

Single-molecule methods reveal enzyme dynamics that cannot be observed with traditional bulk assays. We analyze UvrD helicase translocation on ssDNA using a new technique called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), which has unprecedented spatiotemporal resolution. In SPRNT, a membrane porin MspA in a phospholipid bilayer forms an electrical connection between two salt solutions. A voltage applied across the pore causes ion current to flow through and draws negatively charged DNA bound to UvrD into the pore. UvrD is too large to fit through MspA, and comes to rest on the rim, arresting the DNA's motion. Different DNA nucleotides in the pore modulate the ion current. As UvrD walks along the DNA, it draws the DNA out of the pore and changes the ion current. Therefore, the durations of ion-current states are kinetic measurements of UvrD. Using SPRNT, we show that UvrD moves in single nucleotide steps along DNA and characterize its ATPase activity.