Mechanism of nucleic acid unwinding by SARS-CoV helicase.
Identifieur interne : 001C97 ( Main/Exploration ); précédent : 001C96; suivant : 001C98Mechanism of nucleic acid unwinding by SARS-CoV helicase.
Auteurs : Adeyemi O. Adedeji [États-Unis] ; Bruno Marchand ; Aartjan J W. Te Velthuis ; Eric J. Snijder ; Susan Weiss ; Robert L. Eoff ; Kamalendra Singh ; Stefan G. SarafianosSource :
- PloS one [ 1932-6203 ] ; 2012.
Descripteurs français
- KwdFr :
- MESH :
- enzymologie : Virus du SRAS.
- métabolisme : Acides nucléiques, Helicase.
- Spécificité du substrat.
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : DNA Helicases, Nucleic Acids.
- enzymology : SARS Virus.
- Substrate Specificity.
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.
DOI: 10.1371/journal.pone.0036521
PubMed: 22615777
Affiliations:
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Adedeji</name><affiliation wicri:level="2"><nlm:affiliation>Christopher Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri, School of Medicine, Columbia, Missouri, United States of America.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Christopher Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri, School of Medicine, Columbia, Missouri</wicri:regionArea><placeName><region type="state">Missouri (État)</region></placeName></affiliation></author><author><name sortKey="Marchand, Bruno" sort="Marchand, Bruno" uniqKey="Marchand B" first="Bruno" last="Marchand">Bruno Marchand</name></author><author><name sortKey="Te Velthuis, Aartjan J W" sort="Te Velthuis, Aartjan J W" uniqKey="Te Velthuis A" first="Aartjan J W" last="Te Velthuis">Aartjan J W. Te Velthuis</name></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. Snijder</name></author><author><name sortKey="Weiss, Susan" sort="Weiss, Susan" uniqKey="Weiss S" first="Susan" last="Weiss">Susan Weiss</name></author><author><name sortKey="Eoff, Robert L" sort="Eoff, Robert L" uniqKey="Eoff R" first="Robert L" last="Eoff">Robert L. Eoff</name></author><author><name sortKey="Singh, Kamalendra" sort="Singh, Kamalendra" uniqKey="Singh K" first="Kamalendra" last="Singh">Kamalendra Singh</name></author><author><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. 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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.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Missouri (État)</li></region></list><tree><noCountry><name sortKey="Eoff, Robert L" sort="Eoff, Robert L" uniqKey="Eoff R" first="Robert L" last="Eoff">Robert L. Eoff</name><name sortKey="Marchand, Bruno" sort="Marchand, Bruno" uniqKey="Marchand B" first="Bruno" last="Marchand">Bruno Marchand</name><name sortKey="Sarafianos, Stefan G" sort="Sarafianos, Stefan G" uniqKey="Sarafianos S" first="Stefan G" last="Sarafianos">Stefan G. Sarafianos</name><name sortKey="Singh, Kamalendra" sort="Singh, Kamalendra" uniqKey="Singh K" first="Kamalendra" last="Singh">Kamalendra Singh</name><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. Snijder</name><name sortKey="Te Velthuis, Aartjan J W" sort="Te Velthuis, Aartjan J W" uniqKey="Te Velthuis A" first="Aartjan J W" last="Te Velthuis">Aartjan J W. Te Velthuis</name><name sortKey="Weiss, Susan" sort="Weiss, Susan" uniqKey="Weiss S" first="Susan" last="Weiss">Susan Weiss</name></noCountry><country name="États-Unis"><region name="Missouri (État)"><name sortKey="Adedeji, Adeyemi O" sort="Adedeji, Adeyemi O" uniqKey="Adedeji A" first="Adeyemi O" last="Adedeji">Adeyemi O. Adedeji</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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