INTERACTIONS OF ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASE DNAB PROTEIN WITH NUCLEOTIDE COFACTORS

Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides. The thermo dynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of protein fluorescence and the degree of nucleotide binding, Fluorescence titrations using se veral MANT derivatives of nucleoside diphosphates (MANT-ADP, '-O-(N-me thylantraniloyl)adenosine-5'-diphosphate; MANT-GDP, '-O-(N-methylantra niloyl)guanosine-5'-diphosphate; MANT-CDP, 2'-O-(N-methylantraniloyl)c ytidine-5'-diphosphate; MANT-UDP, ,2'-O-(N-methylantraniloyl)uridine-5 '-diphosphate) have shown that the DnaB helicase has a preference for purine nucleotides, Binding of all modified nucleotides is characteriz ed by similar negative cooperativity, indicating that negative coopera tive interactions are base-independent. Thermodynamic parameters for t he interactions of the unmodified nucleotides (ADP, GDP, CDP, and UDP) and inorganic phosphate (P-i) have been obtained by using the competi tion titration approach, To analyze multiple ligand binding to a finit e circular lattice, for a general case in which each lattice binding s ite can exist in different multiple states, we developed a matrix meth od approach to derive analytical expressions for the partition functio n and the average degree of binding for such cases, Application of the theory to competition titrations has allowed us to extract the intrin sic binding constants and cooperativity parameters for all unmodified ligands. This is the first quantitative estimate of affinities and the mechanism of binding of different unmodified nucleotides and inorgani c phosphate for a hexameric helicase. The intrinsic affinities of all of the studied ATP analogs are lower than the intrinsic affinities of the corresponding ADP analogs, The implications of these results for t he mechanism of helicase action are discussed.