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.

Processivity is the probability that a helicase will move forward along the nucleic acid without dissociating during the unwinding reaction [38, 48] . Results in Figure 4B illustrate that as the length of the duplex increases, the amplitude of the respective unwinding time-course experiments decreases. The processivity of nsp13 was determined by plotting the amplitude against the number of steps with increasing base-pairs in the double-stranded region. The data were fit to equation A = P n . GST-nsp13 unwound nucleic acid with a processivity of 0.8060.03 ( Figure 4C ). The reaction products were separated by thin-layer chromatography and visualized by autoradiography. ATP hydrolysis was quantitated as amount of Pi released and plotted against reaction time. The data were fit to a single exponential equation to calculate initial hydrolysis rates. The derived rates were plotted against ATP concentration and the data points were fit to a hyperbolic function to determine the (A) GST-nsp13 optimal ATP hydrolysis rate k hydro(ATP) 104.164. s 21 and (B) H 6 -nsp13 optimal ATP hydrolysis rate k hydro(ATP) 0.260.006 s 21 (two independent experiments). doi:10.1371/journal.pone.0036521.g002