title: Mechanism of nucleic acid unwinding by SARS-CoV helicase.
abstract: The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.
authors.affiliation: Christopher Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri, School of Medicine, Columbia, Missouri, United States of America.
authors.forename: Adeyemi O
authors.initials: AO
authors.lastname: Adedeji
authors.forename: Bruno
authors.initials: B
authors.lastname: Marchand
authors.forename: Aartjan J W
authors.initials: AJ
authors.lastname: Te Velthuis
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.forename: Susan
authors.initials: S
authors.lastname: Weiss
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
authors.forename: Kamalendra
authors.initials: K
authors.lastname: Singh
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D004265:DNA Helicases / Q000378:metabolism*; D009696:Nucleic Acids / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D009696:Nucleic Acids; D004265:DNA Helicases