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.

To understand the mechanism of nucleic acid unwinding and/ or translocation by nsp13 we need to determine the kinetic parameters associated with the reaction mechanism, such as the unwinding rate, unwinding processivity, directional bias, step size and stoichiometry of ATP coupling [33] . Previously, basic biochemical characterization of nsp13 demonstrated that nsp13 can unwind both double-stranded DNA and RNA in a 59-39 direction, and it can hydrolyze all deoxyribonucleotide and ribonucleotide triphosphates [34, 35] . These studies were conducted using constructs of nsp13 that were fused with maltose binding protein (MBP-nsp13) and hexahistidine (H 6 -nsp13) . Additional studies demonstrated that the amplitude of DNA unwinding by H 6 -nsp13 increases with longer 59-overhang containing DNA substrates [36] . These data advanced our understanding of nsp13 function [37, 38] , but they do not address key mechanistic details such as the kinetic step size, rate of nucleic acid unwinding, and the ATP-coupling stoichiometry. Here, we present a minimal kinetic mechanism of nsp13.