Large T-Antigen Double Hexamers Imaged at the Simian Virus 40 Origin of Replication

Abstract

The initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg²⁺ and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag–DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.

ACKNOWLEDGMENTS

This work was partially supported by grants CAM 07B/0027/1997 from Comunidad de Madrid and BIO98-0761 from Comisión Interministerial de Ciencia y Tecnología to J.M.C. M.V. is recipient of a Postdoctoral Fellowship from Comunidad de Madrid. L.E.D. is supported by a contract from the Ministerio de Educación y Cultura.

We are very grateful to E. Fanning for antibody Pab220 and to O. Llorca for his expert advice and careful reading of the manuscript. The help of Y. Robledo and M. Bárcena is also appreciated. Karen A. Brune is acknowledged for editing the manuscript.