Quantitative analyses of the interactions of the Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA have been performed using the thermodynamically rigorous fluorescence titration technique. This approach allowed us to obtain absolute stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal change and the degree of binding. The analysis of the DnaB helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using a novel spectroscopic Macromolecular Competition Titration (MCT) method developed in the accompanying paper [Jezewska, M. J., & Bujalowski, W. (1996) Biochemistry 35, 2117-2128]. In the presence of the ATP nonhydrolyzable analog AMP-PNP, the DnaB helicase binds polymer DNA with a site-size of 20 +/- 3 nucleotides per protein hexamer. This site-size is independent of the type of nucleic acid base as well as the salt concentration and type of salt. Direct thermodynamic studies of the polynucleotide and oligomer binding to the DnaB hexamer, as well as the competition studies, show that independently of the type of nucleic acid base, as well as salt concentration and type of salt in solution, the helicase has only a single, strong binding site for DNA. Only this site is used when the protein interacts with polymer DNA. Moreover, UV photo-cross-linking experiments with oligonucleotides of different lengths, dT(pT)19, dT(pT)55, and dT(pT)69, suggest that primarily a single subunit of the DnaB helicase hexamer is in contact with the DNA. In interactions with polymer nucleic acids, the DnaB protein shows preferential intrinsic affinity for poly(dA), characterized in our standard conditions (pH 8.1, 10 degrees C, 100 mM NaCl, 5 mM MgCl2) by the intrinsic binding constant K = 6 +/- 2 x 10(6) M-1. These affinities are comparable to the affinities of the single-strand binding proteins in the corresponding solution conditions and strongly suggest that the helicase is capable of binding DNA without additional facilitating factors. Both the intrinsic affinity and the cooperativity are salt dependent. The formation of the DnaB-DNA complex is accompanied by the net release of approximately 2 ions, while another net release of approximately 2 ions accompanies the cooperative interactions. The data indicate an anion effect on the studied interactions and suggests that the released ions most probably originate from both the protein and the nucleic acid. The presence of a single, strong binding site on the hexamer, built of six chemically identical subunits, the very low site-size of the large helicase-DNA complex, and the involvement of a single subunit in contact with the nucleic acid indicate the presence of long-range allosteric interactions in the DnaB helicase which encompass the entire DnaB hexamer. Our sedimentation velocity measurements of the DnaB protein-(AMP-PNP)-5'-fluorescein-(dT)20 ternary complex show that the sedimentation coefficient of the complex is S20,W = 12....

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 thermodynamically 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 several MANT derivatives of nucleoside diphosphates (MANT-ADP, 3',2'-O-(N-methylantraniloyl)adenosine-5'-diphosphate; MANT-GDP, 3',2'-O(N-methylantraniloyl)guanosine-5'-diphosphate; MANT-CDP, 3',2'-O-(N-methylantraniloyl)cytidine-5'-diphosphate; MANT-UDP, 3',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 characterized by similar negative cooperativity, indicating that negative cooperative interactions are base-independent. Thermodynamic parameters for the interactions of the unmodified nucleotides (ADP, GDP, CDP, and UDP) and inorganic phosphate (P(i)) have been obtained by using the competition titration approach. To analyze multiple ligand binding to a finite circular lattice, for a general case in which each lattice binding site can exist in different multiple states, we developed a matrix method approach to derive analytical expressions for the partition function and the average degree of binding for such cases. Application of the theory to competition titrations has allowed us to extract the intrinsic binding constants and cooperativity parameters for all unmodified ligands. This is the first quantitative estimate of affinities and the mechanisms of binding of different unmodified nucleotides and inorganic 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 the mechanism of helicase action are discussed.