STOICHIOMETRY AND MECHANISM OF ASSEMBLY OF SV40 T-ANTIGEN COMPLEXES WITH THE VIRAL ORIGIN OF DNA-REPLICATION AND DNA-POLYMERASE ALPHA-PRIMASE

The interactions of simian virus 40 (SV40) large T antigen with DNA ca rrying the viral origin of DNA replication, as well as its interaction s with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescenc e of 3'(2')-O-(2,4,6-trinitrophenyl)adenosine diphosphate (TNP-ADP) in duced by T antigen binding to the nucleotide was decreased upon bindin g of T antigen to origin DNA. Similarly, the enhanced fluorescence ind uced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon bin ding to human DNA polymerase alpha-primase (pol alpha), but not to rep lication protein A (RPA). Fluorescence titrations revealed noncompetit ive inhibition of TNP-ADP binding by origin DNA, and noncompetitive in hibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able t o bind the TNP nucleotide. From these titrations, we have measured a b inding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral orig in DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier, The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a pref erred ligand over free T antigen in the double hexamer assembly reacti on, preformed T antigen hexamers were incapable of forming double hexa mers on the DNA. The results support a model in which double hexamer a ssembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast wi th this stepwise assembly of T antigen monomers on DNA, hexameric T an tigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these result s for the mechanism of initiation of SV40 DNA replication are discusse d.