Interactions of nucleotide cofactors with both protein components of the Escherichia coli DnaB helicase complex with the replication factor, the DnaC protein, have been examined using MANT-nucleotide analogues. At saturation, in all examined stationary complexes, including the binary, DnaB-DnaC, and tertiary, DnaB-DnaC-ssDNA, complexes, the helicase binds six cofactor molecules. Thus, protein-protein and protein-DNA interactions do not affect the maximum stoichiometry of the helicase-nucleotide interactions. The single-stranded DNA dramatically increases the ATP analogue affinity, while it has little effect on the affinity of the NDP analogues, indicating that stationary complexes reflect allosteric interactions between the DNA- and NTP-binding site prior to the cofactor hydrolysis step and subsequent to product release. In the binary complex, the DnaC protein diminishes the intrinsic affinity and increases the negative cooperativity in the cofactor binding to the helicase; an opposite effect of the protein on the cofactor-helicase interactions occurs in the tertiary complex. The DnaC protein retains its nucleotide binding capability in the binary and tertiary complexes with the helicase. Surprisingly, the DnaC protein-nucleotide interactions, in the binary and tertiary complexes, are characterized by positive cooperativity. The DnaC assembles on the helicase as a hexamer, which exists in two conformational states and undergoes an allosteric transition, induced by the cofactor. Cooperativity of the allosteric transition depends on the structure of the phosphate group of the nucleotide. The significance of the results for the DnaB-DnaC complex activities is discussed.
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