Abstract
Helicases catalyze the unwinding of duplex nucleic acids to aid a variety of cellular processes. Although helicases unwind duplex DNA in the same direction that they translocate on single-stranded DNA, forked duplexes provide opportunities to monitor unwinding by helicase monomers bound to each arm of the fork. The activity of the helicase bound to the displaced strand can be discerned alongside the helicase bound to the translocase strand using a forked substrate with accessible duplexes on both strands labeled with different fluorophores. In order to quantify the effect of protein-protein interactions on the activity of multiple monomers of the Bacteroides fragilis Pif1 helicase bound to separate strands of a forked DNA junction, an ensemble gel-based assay for monitoring simultaneous duplex unwinding was developed (Su et al., 2019). Here, the use of that assay is described for measuring the total product formation and rate constants of product formation of multiple duplexes on a single nucleic acid substrate. Use of this assay may aid characterization of protein-protein interactions between multiple helicase monomers at forked nucleic acid junctions and can assist with the characterization of helicase action on the displaced strand of forked duplexes.