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.

Purified GST nsp13, nsp12H 6 and FMDV 3D pol were treated with DNase I (Fermentas), RNAse A/T1 mix (Fermentas), followed by dialysis against (137 mM NaCl, 1.94 mM K 3 PO 4 , 8.06 mM Na 3 PO 4 , and 2.7 mM KCl, pH 7.4; phosphate buffered saline). GST-nsp13 was incubated with nsp12H 6 or FMDV 3D pol at 4uC for 2 hrs, followed by incubating the mixture with 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was further incubated for 1 hr at 4uC. Precipitates were washed extensively with phosphate buffer saliine. Proteins bound to glutathione beads were eluted using a buffer containg 10 mM Reduced Gluthatione (Sigma Aldrich) and 50 mM Tris pH 8.0 and separated on a SDS-PAGE and purified proteins were visualized by Coomassie-Brilliant Blue staining.