Mechanism of DNA recognition at a viral replication origin

Recognition of the DNA origin by the Epstein- Barr nuclear antigen 1 (EBNA1) protein is the primary event in latent-phase 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(452-641)(.)DNA complex conformers are in slow equilibrium; one dissociates slowly (t(1/2)similar to 41 min) through an undissociated intermediate species and the other corresponds to a fast two-step dissociation route (t(1/2)similar to 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 10(8) M(-1)s(-1), which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent first-order rearrangements of similar to 2.0s(-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.