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.

Similar to previous reports for the MBP-nsp13 and H 6 -nsp13 variants [6, 34, 35] we also found that GST-nsp13 unwinds DNA and RNA substrates containing either a 'fork' or a 59-end singlestranded overhang ( Figure 3B and Figure S1 ). None of the nsp13 variants could unwind substrates with 39-end single-strand overhang or blunt-end DNA and RNA duplexes [6, 34, 35] . However, we show here that nsp13 can unwind nucleic acid secondary structure elements (a stem-loop structure). This was shown using the 48/18-mer and 55/18-mer substrates ( Figure S1 ), which mimic one of the stem loop structures present at the 59 UTR of SARS-CoV genome. These two substrates (48/18-mer and 55/18-mer) differed in the length of 59 single strand overhang before the loop ( Figure 3B and Figure S1 ). The 55/18-mer substrate was readily unwound in contrast to the unwinding of 48/ 18-mer, which was only partially unwound suggesting that nsp13 can unwind stem loops provided the substrate has a 59-end overhang loading region ( Figure 3B ).