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Mol Cell Biol, May 1998, p. 2677-2687, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

Woo S. Joo,dagger Henry Y. Kim, John D. Purviance, K. R. Sreekumar, and Peter A. Bullock*

Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 17 November 1997/Returned for modification 2 January 1998/Accepted 3 February 1998

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

* Corresponding author. Mailing address: Department of Biochemistry A703, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-0447. Fax: (617) 636-6409. E-mail: PBULLOCK{at}OPAL.TUFTS.EDU.

dagger Present address: Graduate Program in Molecular Biology, Sloan-Kettering Cancer Center, New York, NY 10021.

Mol Cell Biol, May 1998, p. 2677-2687, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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Copyright © 1998 by the American Society for Microbiology. All rights reserved.