DNA target recognition mechanism of the Epstein Barr Virus Nuclear Antigen 1 (EBNA1).

ODDO, C.G., FREIRE, E., FRAPPIER, L.AND PRAT GAY, G. DE

Angra dos Reis, R.J., Brasil, 8-12 de noviembre de 2004.

Congreso; First Latin American Protein Society Meeting; 2004

The Protein Society

DNA target recognition mechanism of the Epstein Barr Virus Nuclear Antigen 1 (EBNA1) C. G. Oddo1, E. Freire, L. Frappier2, and G. de Prat Gay1 IIB-Fundacion Leloir, Argentina; 2Department of Medical Genetics and Microbiology, University of Toronto, Canada. The interaction of EBNA1 with DNA plays a crucial role in EBV DNA replication, control of gene expression, and segregation of the EBV viral episome. We investigated the molecular basis of this interaction, using recombinant EBNA1 DNA binding and dimerization domain and synthetic DNA duplexes, through various spectroscopic and biophysical methods. Fluorescence, circular dichroism and gel filtration experiments indicate that the 452-641 domain binds to site 1 DNA duplex in solution with a 1:1 stoichiometry and KD of 0.1 nM, in excellent agreement with previous gel-shift experiments. The interaction shows little dependence on ionic strength from 0.1 to 0.4 M NaCl. In addition, CD monitored titrations suggest a conformational change in the DNA saturating at 1:1 ratio. Isothermal titration calorimetry yields a dissociation constant of 4nM that is close to the value obtained by fluorescence spectroscopic methods, considering the technique concentration range, and a îH of approximately 10 kcal mol-1. Stopped-flow experiments show a second-order binding event of 1.6 x 108 M-1s-1 and a first-order phase with a rate of 2 s-1. Double Jump experiments show that these events are sequential, the binding collision (second order event) is followed by re-arrangement of the protein-DNA complex (first order event). By displacement with excess unlabelled DNA, we determined two phases with off-rates of 0.003 s-1 and 0.0003 s-1. These results provide an insight into the unusual binding interface found in the EBNA1-DNA complex structure and contribute, as a key aspect for its biological function, to the molecular dissection of the binding mechanism.