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dc.contributor.authorParrilla-Doblas, Jara
dc.contributor.authorPonferrada-Marín, María Isabel
dc.contributor.authorRoldán-Arjona, Teresa
dc.contributor.authorRodríguez Ariza, Rafael
dc.date.accessioned2017-12-14T11:48:37Z
dc.date.available2017-12-14T11:48:37Z
dc.date.issued2013
dc.identifier.urihttp://hdl.handle.net/10396/15731
dc.description.abstractActive DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.es_ES
dc.format.mimetypeapplication/pdfes_ES
dc.language.isoenges_ES
dc.publisherOxford University Presses_ES
dc.rightshttps://creativecommons.org/licenses/by-nc-nd/4.0/es_ES
dc.sourceNucleic Acids Research 41(18), 8654–8664 (2013)es_ES
dc.subjectDNA demethylationes_ES
dc.subjectROS1es_ES
dc.subjectProteomicses_ES
dc.titleEarly steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residueses_ES
dc.typeinfo:eu-repo/semantics/articlees_ES
dc.relation.publisherversionhttp://dx.doi.org/10.1093/nar/gkt625es_ES
dc.relation.projectIDGobierno de España. BFU2010- 18838es_ES
dc.relation.projectIDJunta de Andalucía. P07-CVI-02770es_ES
dc.rights.accessRightsinfo:eu-repo/semantics/openAccesses_ES


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