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dc.contributor.authorRuiz-Masó, José A.es_ES
dc.contributor.authorBordanaba-Ruiseco, Lorenaes_ES
dc.contributor.authorSanz, Martaes_ES
dc.contributor.authorMenéndez, Margaritaes_ES
dc.contributor.authorSolar, Gloria deles_ES
dc.date.accessioned2016-09-23T12:43:58Z-
dc.date.available2016-09-23T12:43:58Z-
dc.date.issued2016-09-21-
dc.identifier.citationFrontiers in Molecular Biosciences 3:56 (2016)es_ES
dc.identifier.urihttp://hdl.handle.net/10261/137302-
dc.description15 p.-8 fig.es_ES
dc.description.abstractInitiation of plasmid rolling circle replication(RCR)is catalyzed by a plasmid-encoded Repprotein that performs aTyr-andmetal-dependentsite-specific cleavage of one DNA strand within the double-strand origin(dso)of replication.The crystal structure of RepB,the initiator protein of the streptococcal plasmid pMV158,constitutes the first example of a Repprotein structure from RCR plasmids.It forms a toroidal homohexamericring where each RepBprotomer consists of two domains:theC-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities.Binding of Mn2+ to the active site is essential for the catalytic activity of RepB.In this work,we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches.The analysis of the temperature-induced changes in RepB shows that the first thermal transition,which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host,represents an irreversible conformational change that affects the secondary and tertiary structure of the protein,which becomes prone to self-associate.This transition,which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB,is confined to its N-terminal domain.Mn2+ protects the protein from undergoing this detrimental conformational hange and the ob served protection correlates well with the high-affinity binding of the cation to the active site,as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+ and the Mn2+-driven thermostabilization effect.The level of catalytic activity of the protein,especially in the caseof full-length RepB,cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional,lower-affinity metalbinding site(s),missing in these parate catalytic domain,that must also be saturated for maximal activity.The molecular bases of the thermostabilizing effect of Mn2+ on the N-terminal domain of the protein as well as the potential location of additional metalbinding sitesin the entire RepB are discussed.es_ES
dc.description.sponsorshipThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-70052-R to MM; BFU2010-19597 to GdS, AGL2012-40084-C03 and AGL2015-71923-REDT to GdS). Additional funding to MM was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII).es_ES
dc.language.isoenges_ES
dc.publisherFrontiers Mediaes_ES
dc.relation.isversionofPublisher's versiones_ES
dc.rightsopenAccesses_ES
dc.subjectHUH endonucleaseses_ES
dc.subjectPlasmid-encoded Repproteinses_ES
dc.subjectMetal-dependent catalyticactivity,es_ES
dc.subjectRepB thermostabilityes_ES
dc.subjectMn2+ affinityes_ES
dc.titleMetal-induced stabilization and activation of plasmid replication initiator RepuBes_ES
dc.typeartículoes_ES
dc.identifier.doi10.3389/fmolb.2016.00056-
dc.description.peerreviewedPeer reviewedes_ES
dc.relation.publisherversionhttp://dx.doi.org/10.3389/fmolb.2016.00056es_ES
dc.identifier.e-issn2296-889X-
dc.rights.licensehttp://creativecommons.org/licenses/by/4.0/es_ES
dc.contributor.funderMinisterio de Economía y Competitividad (España)es_ES
dc.contributor.funderInstituto de Salud Carlos IIIes_ES
dc.relation.csices_ES
oprm.item.hasRevisionno ko 0 false*
dc.identifier.funderhttp://dx.doi.org/10.13039/501100003329es_ES
dc.identifier.funderhttp://dx.doi.org/10.13039/501100004587es_ES
dc.identifier.pmid27709114-
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