SV40 large T antigen functions in DNA replication and transformation

Partial Proteolysis of Simian Virus 40 T Antigen Reveals Intramolecular Contacts between Domains and Conformation Changes upon Hexamer Assembly

“…In addition, a role for domain III in contacting origin DNA in the double hexamer was recently reported (34). The proteasesensitive C terminus of Tag harbors a poorly understood host range function needed for virus production in some host cells (1)(2)(3).…”

“…One microgram of Tag per lane was digested with trypsin (t) at a protease/protein mass ratio of 1:500 (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20) and separated by 13.5% SDS-PAGE. Untreated Tag (u) (lanes 3,5,7,9,11,13,15,17,19, and 21) and 10-kDa marker proteins (m) (lane 1) were electrophoresed in parallel. Lanes 1-3 were Coomassie-stained, and lanes 4 -21 were analyzed by Western blotting with monoclonal antibodies whose epitopes in Tag are indicated in the schematic diagram.…”

“…Precipitations were carried out either directly after tryptic cleavage (lanes 2, 3, 6, 7, 10, 11, 14, and 15) or after denaturing the sample (lanes 4, 8, 12, and 16). The products were separated by 13.5% SDS-PAGE and analyzed by immunoblotting with Pab204 (lanes [1][2][3][4][5][6][7][8] or Pab419 (lanes 9 -16). Peptide numbers are indicated on the left, and those that co-precipitated after cleavage, but not after denaturation (den.…”

“…[1][2][3]. In the presence of ATP, Tag monomers bind to specific origin sequences, assembling around the DNA and distorting it locally (reviewed by Refs.…”

“…3, 6 -8). Electron microscopic, biochemical, and genetic studies strongly suggest that the double hexameric complex forms the core of the replisome, leading to the assembly of leading and lagging strand complexes containing a minimum of 10 host proteins (2,3,6,9). Much effort has been aimed at elucidating in detail the operation of this comparatively simple viral replisome with the hope that general principles can be discerned that will increase our understanding of mammalian chromosomal replication.…”