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Molecular and Cellular Biology, January 2000, p. 34-41, Vol. 20, No. 1
Centro Nacional de Biotecnología
(CSIC), Campus de Cantoblanco, 28049 Madrid,
Spain,1 and University of Konstanz
Department of Biology, 78457 Konstanz, Germany2
Received 14 June 1999/Returned for modification 2 August
1999/Accepted 28 September 1999
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.
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Large T-Antigen Double Hexamers Imaged at the
Simian Virus 40 Origin of Replication
*
Corresponding author. Mailing address: Centro Nacional
de Biotecnologia (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-5854543. Fax: 34-91-5854506. E-mail:
carazo{at}cnb.uam.es.
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