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J. Biol. Chem., Vol. 281, Issue 37, 26893-26903, September 15, 2006

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Mechanism of DNA Recognition at a Viral Replication Origin^*

Cristian Oddo, Eleonora Freire, Lori Frappier, and Gonzalo de Prat-Gay¹

From the Instituto Leloir, Patricias Argentinas 435, 1405 Buenos Aires, Argentina and the Department of Medical Genetics and Microbiology, University of Toronto, Ontario M5S 1A8, Canada

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, EBNA1_452–641, 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 EBNA1_452–641·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 (t 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t 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 0.25–1.6 x 10⁸ M^–1 s^–1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of 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.

Received for publication, March 6, 2006 , and in revised form, June 28, 2006.

^* This work was supported by Wellcome Trust Collaborative Research Initiative Grant 066649/Z/01/Z (to G. P.-G.) and by a grant from the Canadian Institutes of Health Research (to L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A Career Investigator from Consejo Nacional de Investigaciones Científicasy Técnicas. To whom correspondence should be addressed. Tel.: 54-0115238-7500; Fax: 54-011-5238-7501; E-mail: gpratgay{at}leloir.org.ar.

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				D. U. Ferreiro, I. E. Sanchez, and G. de Prat Gay Transition state for protein-DNA recognition PNAS, August 5, 2008; 105(31): 10797 - 10802. [Abstract] [Full Text] [PDF]