Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of ∼10 and ∼1000 higher than the value observed for ADP and PO4-. The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the γ phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg2+, to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg2+ cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg2+ has a dual function in nucleotide-helicase interactions. At low [Mg2+], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg2+], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base. © 2005 American Chemical Society.