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 determine whether nsp13 is indeed a 'slow' enzyme, we prepared a GST-nsp13 construct and expressed it in a bacterial The helicase activity of 100 nM nsp13 was measured with 5 nM of each of the substrates at 30uC for 5 secs and the products were separated on a non-denaturing 6% polyacrylamide gel and visualized using a PhosphorImager. C) Four different substrates with 59 overhang lengths varying from 5 to 20 nucleotides were designed to determine the minimum length of loading strand required by nsp13 to efficiently unwind its substrate. The helicase activity of nsp13 (10 nM) was assessed on these substrates (5 nM each) at 30uC for 10 mins and the products were separated on a non-denaturing 6% polyacrylamide gel and visualized using the PhosphorImager. doi:10.1371/journal.pone.0036521.g003 and baculovirus expression systems. GST-nsp13 protein expressed in bacterial systems was insoluble, whereas when expressed in a baculovirus expression system it was highly soluble ( Figure S2 ). GST-nsp13 expressed in baculovirus displayed much higher unwinding and ATPase activities than the H 6 -or MBP-tagged proteins (Figure 1 and Figure 2 ). This enzyme showed same polarity as reported before for two other variants of nsp13 [34, 35] . GST-nsp13 requires at least a five nucleotide 59-end single strand overhang for efficient unwinding similar to human coronavirus 229E helicase [52] . GST-nsp13 can unwind secondary structures of nucleic acid (such as those present in viral genome) as long as a sufficient 59 single strand overhang is available. Unlike MBP-nsp13 and H 6 -nsp13, GST-nsp13 has a helicase activity comparable to other viral, bacterial and eukaryotic helicases. We and others have been unable to hydrolytically remove the affinity tags from nsp13 [34, 35] presumably because the engineered cleavage site is not easily accessible to the protease. Notably, exogenously added GST did not have any effect on the rate of nucleic acid unwinding by both H 6 -nsp13 ( Figures 6C and 6D ) and MBP-nsp13 (data not shown), suggesting that the higher activity of GST-nsp13 is not a result of a GST artifact. It is not clear why GST-nsp13 is more active than H 6 -nsp13. The two proteins appear to have comparable affinities for dsDNA as seen in gel shift experiments (results not shown). Comparison of the ATP hydrolysis rate by GST-nsp13 and H 6 -nsp13 showed that H 6 -nsp13 hydrolyzes ATP slower than GST-nsp13. Hence, based on these experiments we decided to biochemically characterize the GST-nsp13 enzyme, although most of the experiments were also carried out with H 6 -nsp13. Our detailed biochemical analysis showed that GST-nsp13 unwinds DNA at a rate (k u ) of 30 steps per second, with each step being approximately 9.3 bps. The kinetic characterization of several helicases have shown that the step size for DNA unwinding varies between (1-20 bps) [53, 54, 55, 56] . We used Scientist 3.0 (Micromath) software for obtaining the kinetic parameters, and these parameters were also confirmed with the Dynafit 4.0 (BioKin Ltd, MA) and Kintec Explorer (Kintek Corporation, PA) software packages. Helicase processivity provides a measure of the fraction of unwound nucleic acid before dissociation.