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.

Comparison of GST-nsp13, MBP-nsp13, and H 6 -nsp13 helicase and ATPase activities First, we compared the unwinding activities of H 6 -nsp13, GST-nsp13 and MBP-nsp13 using 100 nM of each enzyme and 5 nM 60/40-mer (20ss:40ds) as DNA substrate. The results presented in Figure 1 show that GST-nsp13 unwound half of the substrate in ,0.1 seconds. However, MBP-nsp13 and H 6 -nsp13 reached the same unwinding level at approximately 60 and 100 seconds, respectively. GST-nsp13 unwound the full amount of the nucleic acid in approximately 5 seconds, whereas it took more than 600 seconds for MBP-nsp13 and H 6 -nsp13 to achieve the same task. These results demonstrated that H 6 -nsp13 was the slowest of the three enzymes and that GST-nsp13 is faster than the other variants by up to three orders of magnitude. We also compared the ATPase activities of these three variants of nsp13. We determined ATP hydrolysis by measuring released Pi using thin-layer chromatography and obtained the initial rate constants by fitting the data to single exponential curves. The initial rate constants were then plotted against [ATP] and the data were fit to a hyperbolic function, yielding k hydro(ATP) of 104.164.0 s 21 for GST-nsp13 and 0.2260.006 s 21 for H 6 -nsp13 (Figure 2A and 2B) . The MBP-nsp13 variant also showed similar rate of ATP hydrolysis as the H 6 -nsp13 (data not shown). Hence, most subsequent experiments for biochemical characterization were performed with GST-nsp13, and were generally repeated with H 6 -nsp13 and MBP-nsp13.