title: Mechanism of DNA recognition at a viral replication origin.
abstract: Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.
authors.affiliation: Instituto Leloir, Patricias Argentinas 435, 1405 Buenos Aires, Argentina.
authors.forename: Cristian
authors.initials: C
authors.lastname: Oddo
authors.forename: Eleonora
authors.initials: E
authors.lastname: Freire
authors.forename: Lori
authors.initials: L
authors.lastname: Frappier
authors.forename: Gonzalo
authors.initials: G
authors.lastname: de Prat-Gay
mesh_terms: D000818:Animals; D016880:Anisotropy; D002151:Calorimetry; D002460:Cell Line; D004279:DNA, Viral / Q000737:chemistry*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D018741:Replication Origin; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; C417029:EBV-encoded nuclear antigen 1