The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.
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.
Dean
F B
FB
Program in Molecular Biology, Sloan-Kettering Cancer Center, New York, New York 10021.
Borowiec
J A
JA
Eki
T
T
Hurwitz
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016367:Baculoviridae; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008957:Models, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism / Q000648:ultrastructure; D013539:Simian virus 40 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate