Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

Adedeji, Adeyemi O.; Marchand, Bruno; te Velthuis, Aartjan J. W.; Snijder, Eric J.; Weiss, Susan; Eoff, Robert L.; Singh, Kamalendra; Sarafianos, Stefan G.

ATP hydrolysis measurements were carried out under single turnover conditions similar to those used in unwinding assays. 100 nM GST-nsp13 and 5 nM DNA substrates were loaded into one sample loop (15 ml) and various concentrations of c -32 P-ATP (2, 5, 12, 25, or 50 mM), together with 1 mM unlabeled 18-mer DNA (to prevent re-annealing on the unwound labeled strand) were loaded into the other sample loop (15 ml). Both samples contained a reaction buffer consisting of 20 mM HEPES pH 7.5, 20 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/ml BSA, and 5% glycerol. Samples were rapidly mixed at 30uC and reactions were allowed to proceed for desired time periods ranging between 0.005 to 0.5 s for GST-nsp13 or 0.005 to 5 s for H 6 -nsp13 and MBP-nsp13 prior to quenching with 100 mM EDTA, 0.2% SDS, and 20% glycerol. Reaction products were separated by thin-layer chromatography on polyethyleneimine-cellulose F plates (Merck) using 0.5 M LiCl as the liquid phase and visualized by autoradiography. The monophosphate (Pi) hydrolysis product was quantitated using ImageQuant (Amersham) and used to calculate hydrolyzed fraction of ATP. Experiments were per-formed twice and kinetic parameters were determined using the Prism (GraphPadInc) software. Figure S1 Oligonucleotides and substrates used in this study. The Cy3-labeled strands are marked by asterisks. The sequences in red are self-annealing within the longer strand, while the green sequences denote the complementary sequences in the two strands. Figure S4 Interaction of GST-nsp13 and nsp12H 6 . A) Purified GST nsp13, nsp12H 6 and FMDV 3D pol were dialyzed against (137 mM NaCl, 1.94 mM K 3 PO 4 , 8.06 mM Na 3 PO 4 , and 2.7 mM KCl, pH 7.4; phosphate buffered saline). GST-nsp13 was incubated with nsp12H 6 or FMDV 3D pol at 4uC for 12 hrs, followed by incubating the mixture with 50% slurry of glutathioneconjugated Sepharose beads (Amersham Biosciences), and the binding reaction was further incubated for 4 hrs at 4uC. Precipitates were washed extensively with phosphate buffer saliine. Proteins bound to glutathione beads were eluted and separated on a SDS-PAGE and purified proteins were visualized by Coomassie-Brilliant Blue staining. The left and right panels represent the SDS-PAGE for GSTnsp13-nsp12 interaction and the GST-nsp13/FMDV 3D pol data respectively. B) Binding of 60/40mer (20ss:40ds) DNA substrates, with GST-nsp13 and varying concentrations of nsp12H 6 was assessed using a gel mobility shift assay. Samples were analysed on a 5% non-denaturing polyacrylamide gel. (TIF)