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.

The assay conditions for single turnover helicase activity measurements were similar to those used for initial characterization except that the concentration of nsp13 was in excess over the DNA substrates. The reactions were carried out in 20 mM HEPES (pH 7.5), 20 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/ml BSA, and 5% glycerol at 30uC using a Rapid Chemical Quench Flow instrument (KinTek Corp.) [43, 57] . GST-nsp13 (100 nM) and DNA substrates (5 nM) were loaded into one of the sample loops (15 ml), whereas ATP (2 mM), and unlabeled DNA substrate (1 mM) were loaded into the other sample loop (15 ml). Samples were rapidly mixed and the reaction was quenched with 100 mM EDTA, 0.2% SDS, and 20% glycerol after desired time intervals (5 ms to 1 s). Reaction products were resolved and quantitated as described above. Experiments were performed at least three times. To determine the effect of nsp12 (SARS-CoV RdRp) on the helicase activity of nsp13, the experiment was as described above except that varying concentrations of nsp12-H 6 , GST-nsp13 (100 nM) and 59-Cy3-labeled 80/60-mer (20ss:60ds) DNA (5 nM) were loaded together into one of the sample loops.