Characterization of the Nucleotide Binding Properties of SV40 T Antigen Using Fluorescent 3‘(2‘)-O-(2,4,6-Trinitrophenyl)adenine Nucleotide Analogues

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Abstract
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, 55−85]. To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3‘(2‘)-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues. Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission 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 bound to the ATP binding site with dissociation constants of 0.35 μM and 2.6 μM. 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 more temperature dependent than that of TNP-ADP. The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP were 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 the affinity of various nucleotides and analogues for T antigen. The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.