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 reports have shown that several DNA and RNA helicases interact with their corresponding polymerases. These interactions generally enhance the unwinding activity of helicases [49, 50, 51] . Evidence from yeast-two-hybrid experiments suggested that SARS-CoV polymerase (nsp12) interacts with nsp13 [31] . To investigate if nsp12 affects the helicase activity of nsp13 we monitored the unwinding of the 80/60-mer DNA substrates (5 nM) by GST-nsp13 (100 nM) in the presence of nsp12 (0 nM, 250 nM, and 500 nM) under conditions described in 'Materials and Methods'. The results showed that 500 nM nsp12 clearly enhanced the unwinding rate (mk U , base-pairs per second) of nsp13 by ,2-fold ( Figure 5 and Table 3 ). This enhancement is accompanied by a change in the step size from 7.9 to 14.3 for this specific substrate (Table 3) demonstrating that the presence of nsp12 can change the rate and the mechanism of helicase activity. 500 nM of nsp12 was used because optimal unwinding of nucleic acid by nsp13 in the presence of nsp12 was observed at this concentration ( Figure S3 ). Also, the effect of nsp12 on the ATPase activity of nsp13 was examined, and no enhancement of the ATPase activity was observed (data not shown).Our data suggest that this enhancement is independent of the type of substrate (dsDNA vs. dsRNA) or protein tag (GST vs. H 6 ). A similar effect was observed when H 6 -nsp13 helicase activity was monitored in the presence of nsp12. The rate of unwinding the shorter 31/18mer RNA substrate increased from 0.360.02 to 1.560.15 (mk U , base-pairs per second) and the rate of unwinding 31/18-mer DNA increased from 0.760.05 to 1.860.07 (mk U , base-pairs per second) ( Figures 6C,6D and Table 4 ) with minimal change in the amplitude. However, the effect on the amplitude is more significant with the substrate with longer duplex region, 80/60mer DNA substrate ( Figure 5 ). Using the same 31/18-mer substrates, GST-nsp13 exhibited similar helicase activity enhancement as H 6 -nsp13 ( Figures 6A, 6B , and Table 4 ). Notably, this enhancement is specific, as it was not observed when the nsp12 SARS RdRp was substituted with the foot-and-mouth disease virus RdRp (FMDV 3Dpol) ( Figures 6A, 6B , and Table 4 ).