Fluorescent Biosensor-Based Real-Time Helicase Assay

Real-time helicase assays were performed using a fluorescent biosensor for ssDNA based on the Plasmodium falciparum SSB protein (fSSB; Chisty et al., 2018 (link)). Reactions were performed in RecBCD buffer (25 mM Tris-HCl pH 7.5, 10 mM NaCl, 6 mM MgCl₂, 0.1 mg/ml BSA) supplemented with 10 pM RecBCD, 1 uM (in ntds; ~10 pM molecules) bacteriophage lambda DNA (New England Biolabs), 25 nM fSSB (tetramer) and gp5.9 at the stated concentration. After a 10 min pre-incubation, reactions were initiated by the addition of 2 mM ATP. Fluorescence intensity was monitored using a Cary Eclipse Fluorescence Spectrophotometer (excitation wavelength 430 nm, emission wavelength 475 nm, excitation and emission slit widths of 10 nm and 5 nm, respectively). Assays were performed in triplicate, and the initial rates reported are the mean and standard error for the three repeats. To obtain IC₅₀ values for inhibition of RecBCD by gp5.9, data describing the initial unwinding rate as a function of log₁₀[gp5.9] were fit to the sigmoidal dose–response equation of GraphPad Prism. $\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\mathrm{r}}_{\mathrm{m}\mathrm{i}\mathrm{n}}+({\mathrm{r}}_{\mathrm{m}\mathrm{a}\mathrm{x}}-{\mathrm{r}}_{\mathrm{m}\mathrm{i}\mathrm{n}})/(1+{10}^{\wedge }(\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{I}\mathrm{C}50-\mathrm{l}\mathrm{o}\mathrm{g}[\mathrm{g}\mathrm{p}5.9]))$
r_min and r_max are the minimum and maximum DNA unwinding rates, respectively. Values for the unwinding rates were normalised to 100% for a zero gp5.9 control, and so for the purposes of the fits the values for r_max and r_min were constrained to 100 and 0 respectively.