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.

In order to estimate the kinetic step size of the SARS-CoV helicase we performed single-turnover assays by pre-incubating excess of GST-nsp13 with partial-duplex substrates. The change in the fraction of unwound ssDNA product with time is shown in Figure 4B . The experimental data for all the substrates were fit globally to equation (1) derived from Scheme 1 ( Figure 4A ). The resulting fits are shown in Figure 4B and the kinetic parameters are listed in Table 1 . Notably, the amplitude of product decreases as the length L T of the dsDNA substrate increases, consistent with a dissociation step as shown in Scheme 1 ( Figure 1 ). The individual uncorrected kinetic step size for different lengths of dsDNA varied from 14.3 to 16.4 as the length of dsDNA region decreased from 60 to 18 bp. Previous studies have shown that the last 8-10 bp of dsDNA can melt spontaneously in the presence of an active helicase [38] . Thus, it is essential to take into consideration the length of dsDNA that spontaneously melts when calculating the kinetic step size rather than using the full length of dsDNA. Hence, the value L 0 used in Table 2 and equation 7 was set to 10 bp.