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.

Helicase activity was measured by incubating 100 nM GST-nsp13 with 5 nM 60/40-mer DNA substrate ( Figure S1 ) in a reaction buffer containing 20 mM HEPES pH 7.5, 20 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA, 5 mM MgCl 2 , and 2 mM ATP at 30uC for various times [34] . The reaction mixture also contained 1 mM unlabeled 40-mer DNA as trap. Reactions were quenched by the addition of equal volume of loading buffer (100 mM EDTA, 0.2% SDS and 20% glycerol). Unless otherwise mentioned, reactant concentrations refer to the final concentration in the reaction mixture. The released single-stranded DNA (ssDNA) product and unwound double-stranded DNA (dsDNA) were resolved on a 6% non-denaturing-PAGE (polyacrylamide gel electrophoresis) using a running buffer containing 89 mM Tris-Borate pH 8.2, and run for 2 hours at 4uC and 150 V. The controls for measuring maximum unwinding were dsDNA denatured by heating for 5 min at 95uC and loading immediately on the gel as suggested by Ahnert et al. [57] . In this and subsequent assays the gels were scanned with a phosphorimager (FLA 5000, FujiFilm). The band intensities representing ssDNA and dsDNA were quantitated using the ImageQuant software (Pharmacia). The fraction of unwound DNA was plotted against time and the kinetic parameters described in the mechanism were determined by non-linear regression using the Graphpad Prism (GraphPad Inc.) and Scientist (Micromath) programs.