Single-Molecule Study of Ded1 Helicases using a Hairpin Substrate

Abstract

DEAD-box helicases remodel RNA structures, DNA/RNA hybrids and RNA-protein complexes that are essential in gene regulation. So far their mechanism and functioning is very much debated and the classical or bulk assays are not sufficient to answer these questions. We have developed a new simple magnetic tweezers based single molecule cyclic assay, acting in parallel on tens of molecules at the same time, which allows detecting the unwinding of short RNA/DNA hybrids. Comparing this information with and without an interacting agent can present some insights about the agent. We implement our assay to study DEAD-box helicase Ded1 which is known to only melt a few bases of DNA/RNA duplex. The observation of unwinding by Ded1 helicase in the presence of ATP indicates that the enzyme melt the duplex rather than translocating along ssDNA. Although DEAD-box proteins are most active with substrates containing single-stranded overhangs, they are also able to unwind short blunt-end duplexes. This has led to the proposal that DEAD-box proteins directly interact with the duplex, but they are “activated” by single-stranded RNA bound at another site. Moreover, it has been proposed that the proteins work by localized destabilization of the RNA helical regions. Hence, the proteins lack processivity. We performed the helicase activity measurement at various concentrations of proteins. Our results show that Ded1 activity is maximum in case of RNA oligo with 5’ overhang. The helicase activity is observed only in presence of ATP while the ATP analogues supported annealing activity. We have also confirmed that the enzyme has no visible effect on the unwinding of DNA.