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.

Previous studies have shown that some helicases exhibit a clear preference for either RNA or DNA [11, 44, 45, 46, 47] . To determine if GST-nsp13 prefers one substrate over the other, we designed 38/18-mer DNA and RNA duplexes ( Figure S1 ) of the same sequence and monitored their unwinding rates under singleturnover conditions. The time course of DNA and RNA Figure 1 . Comparison of unwinding activity of three nsp13 variants. Comparison of the helicase activity at varying time points for GST-nsp13 (Glutathione S-Transferase-tagged nsp13), MBP-nsp13 (Maltose Binding Protein-tagged nsp13), and H 6 -nsp13 (hexahistidine-tagged nsp13) using 100 nM of each enzyme and 5 nM 60/40-mer (30ss:30ds) as substrate at 30uC. The products were separated and analyzed by 6% nondenaturing PAGE. Time points for the GST-nsp13-catalyzed unwinding experiments are more than 100 times smaller than for the MBP-nsp13and H 6 -nsp13-catalyzed experiments (0.01-5 vs. 5-600 seconds). doi:10.1371/journal.pone.0036521.g001 unwinding in Figure 3A demonstrates that GST-nsp13 does not exhibit a significant preference for either RNA or DNA substrates. A similar lack of preference was also observed for MBP-nsp13 and H 6 -nsp13 (data not shown) and was consistent with previous reports for these two enzymes [34, 35] . Henceforth, most experimental analysis was carried out using DNA substrates.