RecQ helicases are essential for cell viability and replication. These helicases are capable of translocation on single-stranded DNA and unwinding of double-stranded DNA by using the chemical energy from ATP hydrolysis. Mutations of RecQ helicases are implicated in a number of genetic disorders, while others have important roles in the development or treatment of cancer. The Werner’s syndrome helicase (WRN) is a notable human RecQ helicase that maintains the genome by its unwinding complex present in telomeres, such as Holiday junctions and G-quadruplexes. These unique abilities make WRN a useful drug target for cancers with microsatellite instability (MSI), since these cancers rely on WRN to survive. RecQ enzymes have been studied using single-molecule techniques such as molecular tweezers and AFM. However, the resolution of these techniques is not sufficient to observe the ∼1 ms-long, sub-nanometer motions of the helicase are not met by these techniques. In nanopore tweezers (NTs)—a new single-molecule—negatively charged DNA conjugated to a motor enzyme is drawn into a nanometer-scale pore protein by an electric field. As the helicase steps along the DNA sequence, the DNA moves through the pore, changing the conductance and thereby enabling measurements of the motor enzyme at a resolution capable of resolving individual kinetic substrates of motor enzymes walking on DNA/RNA. Here, we developed an NT assay for the WRN helicase. We found an ATP-dependent kinetic substrate within the WRN ATPase cycle, similar to stepping patterns found in Hel308 and RecQ1 helicases. Our findings pave the way to deeper insights into WRN’s mechanochemical properties and shed light on the mechanisms by which small molecules could affect WRN. Understanding the detailed molecular origins of WRN kinetics will enable rational drug design against MSI cancer.
Saad et al. (2026) studied this question.