CHARACTERIZATION OF THE NUCLEOTIDE-BINDING PROPERTIES OF SV40 T-ANTIGEN USING FLUORESCENT 3'(2')-O-(2,4,6-TRINITROPHENYL)ADENINE NUCLEOTIDEANALOGS

ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA [Fanning, E., and Knippers, R. (1992) Annu. Rev, Biochem. 61, 55-8 5]. To better explore the functions of T antigen during the replicatio n process, we have studied the interactions of T antigen with fluoresc ent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues . Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluoresc ence enhancement and a concomitant blue shift (11 nm) of the maximal e mission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50%. Both signals were utilized to characterize the nucleotide binding activity of T antigen. TNP-ATP and TNP-ADP boun d to the ATP binding site with dissociation constants of 0.35 mu M and 2.6 mu M. TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold. The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen. The binding of TNP-ATP was m ore temperature dependent than that of TNP-ADP. The enthalpy change co ntributed nearly half of the energy for TNP-ATP binding, whereas bindi ng of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP w ere strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site. Like the parent nucleotides, they also induced T antigen hexamer formation . Using the TNP nucleotides as fluorescent probes, we have measured th e affinity of various nucleotides and analogues for T antigen. The res ults indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggestin g closely related ATP binding sites in the three DNA helicases.