Mechanism of nucleic acid unwinding by SARS-CoV helicase
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 func...
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| creator | Adedeji, Adeyemi O Marchand, Bruno Te Velthuis, Aartjan J W Snijder, Eric J Weiss, Susan Eoff, Robert L Singh, Kamalendra Sarafianos, Stefan G |
| description | 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_str_mv | 10.1371/journal.pone.0036521 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0036521</identifier><identifier>PMID: 22615777</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Acids ; Biochemistry ; Biology ; Catalysis ; Coronaviridae ; Coronaviruses ; Deinococcus radiodurans ; Deoxyribonucleic acid ; DNA ; DNA helicase ; DNA Helicases - metabolism ; DNA-directed RNA polymerase ; Double-stranded RNA ; Energy consumption ; Energy use ; Enzymes ; Foot & mouth disease ; Gene expression ; Health aspects ; Hepatitis ; Hydrolysis ; Immunology ; Infectious diseases ; Intermediates ; Laboratories ; Life sciences ; Medicine ; Nucleic acids ; Nucleic Acids - metabolism ; Polarity ; Polymerase ; Proteins ; Ribonucleic acid ; RNA ; RNA helicase ; RNA synthesis ; RNA-directed RNA polymerase ; SARS Virus - enzymology ; Severe acute respiratory syndrome ; Substrate Specificity ; Transcription ; Unwinding ; Virology ; Viruses</subject><ispartof>PloS one, 2012-05, Vol.7 (5), p.e36521-e36521</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 Adedeji et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Adedeji et al. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-198eda8a46da47498322cd5cd00dc6cfead55cf42a9ce50cf2a866ba558ddba23</citedby><cites>FETCH-LOGICAL-c692t-198eda8a46da47498322cd5cd00dc6cfead55cf42a9ce50cf2a866ba558ddba23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352918/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352918/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22615777$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Adedeji, Adeyemi O</creatorcontrib><creatorcontrib>Marchand, Bruno</creatorcontrib><creatorcontrib>Te Velthuis, Aartjan J W</creatorcontrib><creatorcontrib>Snijder, Eric J</creatorcontrib><creatorcontrib>Weiss, Susan</creatorcontrib><creatorcontrib>Eoff, Robert L</creatorcontrib><creatorcontrib>Singh, Kamalendra</creatorcontrib><creatorcontrib>Sarafianos, Stefan G</creatorcontrib><title>Mechanism of nucleic acid unwinding by SARS-CoV helicase</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Acids</subject><subject>Biochemistry</subject><subject>Biology</subject><subject>Catalysis</subject><subject>Coronaviridae</subject><subject>Coronaviruses</subject><subject>Deinococcus radiodurans</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA helicase</subject><subject>DNA Helicases - metabolism</subject><subject>DNA-directed RNA polymerase</subject><subject>Double-stranded RNA</subject><subject>Energy consumption</subject><subject>Energy use</subject><subject>Enzymes</subject><subject>Foot & mouth disease</subject><subject>Gene expression</subject><subject>Health aspects</subject><subject>Hepatitis</subject><subject>Hydrolysis</subject><subject>Immunology</subject><subject>Infectious diseases</subject><subject>Intermediates</subject><subject>Laboratories</subject><subject>Life sciences</subject><subject>Medicine</subject><subject>Nucleic acids</subject><subject>Nucleic Acids - metabolism</subject><subject>Polarity</subject><subject>Polymerase</subject><subject>Proteins</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA helicase</subject><subject>RNA synthesis</subject><subject>RNA-directed RNA polymerase</subject><subject>SARS Virus - enzymology</subject><subject>Severe acute respiratory syndrome</subject><subject>Substrate Specificity</subject><subject>Transcription</subject><subject>Unwinding</subject><subject>Virology</subject><subject>Viruses</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkluLEzEYhgdR3IP-A9EBYdGL1pxn5kYoxUNhZWGrexu-yaFNmSbdyYy6_97Uzi4d2QvJRULyfO93yJtlrzCaYlrgD5vQtx6a6S54M0WICk7wk-wUV5RMBEH06dH5JDuLcYMQp6UQz7MTQgTmRVGcZuU3o9bgXdzmwea-V41xKgfldN77X85r51d5fZcvZ9fLyTzc5GvTOAXRvMieWWiieTns59mPz5--z79OLq--LOazy4kSFekmuCqNhhKY0MAKVpWUEKW50ghpJZQ1oDlXlhGolOFIWQKpxBo4L7WugdDz7M1Bd9eEKIemo8SUMUow4iIRiwOhA2zkrnVbaO9kACf_XoR2JaHtXOpMFoKX2KqqAo1YiXileZ0KqUhNqWWWJa2PQ7a-3hqtjO9aaEai4xfv1nIVfkpKOalwmQTeDQJtuO1N7OTWRWWaBrwJfaobYc5EUXCe0Lf_oI93N1ArSA04b0PKq_aicsaKAqWB0r3W9BEqLW22TiWHWJfuRwHvRwGJ6czvbgV9jHKxvP5_9upmzF4csWsDTbeOoek7F3wcg-wAqjbE2Br7MGSM5N7g99OQe4PLweAp7PXxBz0E3Tua_gGFB_PA</recordid><startdate>20120515</startdate><enddate>20120515</enddate><creator>Adedeji, Adeyemi O</creator><creator>Marchand, Bruno</creator><creator>Te Velthuis, Aartjan J W</creator><creator>Snijder, Eric J</creator><creator>Weiss, Susan</creator><creator>Eoff, Robert L</creator><creator>Singh, Kamalendra</creator><creator>Sarafianos, Stefan G</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PHGZM</scope><scope>PHGZT</scope><scope>PIMPY</scope><scope>PJZUB</scope><scope>PKEHL</scope><scope>PPXIY</scope><scope>PQEST</scope><scope>PQGLB</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20120515</creationdate><title>Mechanism of nucleic acid unwinding by SARS-CoV helicase</title><author>Adedeji, Adeyemi O ; Marchand, Bruno ; Te Velthuis, Aartjan J W ; Snijder, Eric J ; Weiss, Susan ; Eoff, Robert L ; Singh, Kamalendra ; Sarafianos, Stefan G</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-198eda8a46da47498322cd5cd00dc6cfead55cf42a9ce50cf2a866ba558ddba23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Acids</topic><topic>Biochemistry</topic><topic>Biology</topic><topic>Catalysis</topic><topic>Coronaviridae</topic><topic>Coronaviruses</topic><topic>Deinococcus radiodurans</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA helicase</topic><topic>DNA Helicases - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22615777</pmid><doi>10.1371/journal.pone.0036521</doi><tpages>e36521</tpages><oa>free_for_read</oa></addata></record> |
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| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Acids Biochemistry Biology Catalysis Coronaviridae Coronaviruses Deinococcus radiodurans Deoxyribonucleic acid DNA DNA helicase DNA Helicases - metabolism DNA-directed RNA polymerase Double-stranded RNA Energy consumption Energy use Enzymes Foot & mouth disease Gene expression Health aspects Hepatitis Hydrolysis Immunology Infectious diseases Intermediates Laboratories Life sciences Medicine Nucleic acids Nucleic Acids - metabolism Polarity Polymerase Proteins Ribonucleic acid RNA RNA helicase RNA synthesis RNA-directed RNA polymerase SARS Virus - enzymology Severe acute respiratory syndrome Substrate Specificity Transcription Unwinding Virology Viruses |
| title | Mechanism of nucleic acid unwinding by SARS-CoV helicase |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-21T20%3A27%3A31IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Mechanism%20of%20nucleic%20acid%20unwinding%20by%20SARS-CoV%20helicase&rft.jtitle=PloS%20one&rft.au=Adedeji,%20Adeyemi%20O&rft.date=2012-05-15&rft.volume=7&rft.issue=5&rft.spage=e36521&rft.epage=e36521&rft.pages=e36521-e36521&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0036521&rft_dat=%3Cgale_plos_%3EA477083235%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1344321056&rft_id=info:pmid/22615777&rft_galeid=A477083235&rft_doaj_id=oai_doaj_org_article_76581fc99ad048059d5b00d92b33f4f4&rfr_iscdi=true |