Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins

In the last 15 years, single molecule techniques have allowed us to observe the transient, heterogeneous, and multi‐step behavior of biomolecules often averaged out and lost in ensemble assays. Helicases form a ubiquitous class of enzymes that function in many aspects of nucleic acid metabolism which is central to life. We used single molecule Förster resonance energy transfer (FRET) and optical tweezers force spectroscopy to study translocation and unwinding mechanism of Escherichia coli Rep, a model helicase with 3’5’ motor translocation activity on single‐stranded DNA (ssDNA) fueled by ATP hydrolysis. When a translocating Rep on a 3’‐overhang DNA reaches the DNA duplex junction, instead of dissociating, it snaps back to the 3’ end, restarting another shuttling cycle. We investigated the mechanism of repetitive shuttling and discovered the same behavior on various DNA substrates. We concluded that the repetitions are induced by the lack of ssDNA track ahead of the Rep. Using the repetitive shuttling assay, we explored the translocation mechanism of Rep in detail. First, we perturbed the ssDNA binding network via biochemical mutations that led to determination of the key residues that control shuttling speed, ATPase activity, and directionality. Second, we tested the effects of the DNA lesions on Rep translocation, observing that the irregularities encompassing 1‐3 nucleotides in the backbone and nucleobases only caused transient stalls. To probe the unwinding mechanism, we developed a conformational control assay which turned wild type Rep monomers with no detectable DNA unwinding activity into super‐helicases (Rep‐X) via internal covalent crosslinking. Rep‐X can unwind thousands of base pairs processively even against large forces. We also showed that partner proteins of a similar enzyme turn on the unwinding activity by stabilizing the active form. Lastly, the orientation dependence of FRET is experimentally shown between nucleic acid conjugated cyanine fluorophores that constitutes the first demonstration of this effect in a biophysical system since its formulation by Theodor Förster in 1948