Quantitative and accurate analyses of protein-nucleic acid interactions in solution are greatly facilitated if the formation of the complex is accompanied by a large change of the spectroscopic signal (e.g., fluorescence) originating from the protein or nucleic acid. However, there are many instances when protein-nucleic acid interactions do not induce adequate changes in spectroscopic properties of the interacting macromolecules. We describe the theoretical and experimental aspects of a general method to analyze such protein-nucleic acid interactions. The method is based on quantitative titrations of a reference nucleic acid with the protein in the presence of a competing nucleic acid whose interaction parameters with the protein are to be determined. The Macromolecule Competition Titration (MCT) method allows for the determination of the absolute average binding density and the free protein ligand concentration over a large binding density range, unavailable by other methods, and construction of a model-independent true binding isotherm. Moreover, the determination of the absolute binding density of the ligand on nonfluorescent nucleic acid is independent of a priori knowledge of the binding characteristics of the protein to the reference fluorescent nucleic acid. Although the MCT method is applicable to any type of physicochemical signal that can be used to monitor the binding, we discuss the details of the method as it applies to the analysis monitored by a change in the nucleic acid fluorescence intensity and anisotropy upon binding a ligand. Moreover, the interaction parameters for a given nucleic acid can be determined by using as a reference the long polymer nucleic acid as well as short oligomers. In particular, the analysis is greatly simplified if the short fluorescent nucleic acid fragment, spanning the exact site-size of the complex and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. We have illustrated the MCT method by applying it to the binding of the Escherichia coli DnaB helicase to unmodified, nonfluorescent single-stranded nucleic acids where the interactions are not accompanied by any adequate spectroscopic signal changes. In order to analyze simultaneous binding of a ligand to different competing nucleic acid lattices, we introduced the combined application of the McGhee-von Hippel theory and the Epstein combinatorial approach for the binding of a large ligand to a linear, homogeneous nucleic acid lattice. Our approach allows one to perform a direct fit of the entire experimental isotherm for the protein binding to two competing nucleic acid lattices without resorting to complex numerical calculations.

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....

The interactions of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA have been studied using the thermodynamically rigorous fluorescence titration technique, which 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. Binding of the DnaB protein to the ssDNA fluorescent derivative poly(d epsilon A) is accompanied by a strong increase of the nucleic acid fluorescence. We show that, in the presence of the ATP nonhydrolyzable analog AMP-PNP, the DnaB helicase binds polymer ssDNA with the site-size of 20 +/- 3 nucleotides per protein hexamer. This stoichiometry has been fully confirmed in the binding experiments with ssDNA oligomers of 40 and 20 residues in length. Two DnaB hexamers bind to 40-mer, and one DnaB hexamer binds to 20-mer. Thermodynamic studies of the 20-mer binding to the DnaB hexamer show that the hexamer has a single, strong binding site for ssDNA. Moreover, photo-cross-linking experiments indicate that only a single subunit is primarily in contact with ssDNA. This surprisingly very low site-size of the large hexameric helicase--ssDNA complex, the existence of only a single, strong ssDNA binding site on the hexamer, and the results of photo-cross-linking experiments preclude the possibility of extensive wrapping of the ssDNA around the hexamer and formation of the complex in which all six protomers are simultaneously bound to ss nucleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARYAnalyses of interactions of the E. coli replicative helicase, PriA protein, with a singlestranded DNA have been performed, using the quantitative fluorescence titration technique.The stoichiometry of the PriA helicase -ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriAssDNA complex, i.e., the maximum number of nucleotide residues occluded by the PriA helicase in the complex is 20 ± 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of ~6 nucleotides on both sides of the binding site.

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.