The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin

• Published in: The Journal of biological chemistry 1992-07, Vol.267 (20), p.14129-14137
• Main Authors: DEAN, F. B, BOROWIEC, J. A, EKI, T, HURWITZ, J
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Animals; Antigens, Polyomavirus Transforming - genetics; Antigens, Polyomavirus Transforming - metabolism; Antigens, Polyomavirus Transforming - ultrastructure; Baculoviridae; Biological and medical sciences; DNA Replication; DNA, Viral - genetics; DNA, Viral - metabolism; Fundamental and applied biological sciences. Psychology; Insecta; Kinetics; Macromolecular Substances; Microscopy, Electron; Models, Genetic; Molecular and cellular biology; Molecular genetics; Protein Binding; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Replication; Simian virus 40 - genetics; Simian virus 40 - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)49688-9

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.