Maria J. Jezewska

Does Single-stranded DNA Pass through the Inner Channel of the Protein Hexamer in the Complex with the Escherichia coli DnaB Helicase? FLUORESCENCE ENERGY TRANSFER STUDIES

The structure of the complex of theEscherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 Å) is the same as in the complex with the isolated ssDNA oligomer (R = 47 Å) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.

Flexibility of the rings: Structural asymmetry in the DnaB hexameric helicase

DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding. Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain. Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used. These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.

Interactions of the Escherichia coli DnaB Helicase Hexamer with the Replication Factor the DnaC Protein. Effect of Nucleotide Cofactors and the ssDNA on Protein–Protein Interactions and the Topology of the Complex

Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and fluorescence energy transfer techniques. The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding. Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB-DnaC complex formation. The DnaC binding to the unmodified helicase has been characterized in competition experiments. The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer. The positive cooperative interactions are limited to the two neighboring DnaC molecules. Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(+/-0.5)x10(5)M(-1) and cooperativity parameter sigma=21+/-5. These data suggest strongly that the DnaC-DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution. The DnaC nucleotide-binding site is not involved in the stabilization of the complex. Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex. The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding. However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC-DnaB-ssDNA, formed in the presence of AMP-PNP as compared to ADP. The topology of the ternary complex DnaC-DnaB-ssDNA has been determined using the fluorescence energy transfer method. In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase. The significance of the results in the functioning of the DnaB helicase-DnaC protein complex is discussed.

Functional and Structural Heterogeneity of the DNA Binding Site of the Escherichia coli Primary Replicative Helicase DnaB Protein

The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite. Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.

Binding Specificity of Escherichia coli Single-Stranded DNA Binding Protein for the χ Subunit of DNA pol III Holoenzyme and PriA Helicase

The Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions, we have used isothermal titration calorimetry to examine the thermodynamics of binding of SSB to two E. coli proteins important for DNA replication, the chi subunit of DNA polymerase III holoenzyme and the PriA helicase. We find that an SSB tetramer can bind up to four molecules of either protein primarily via interactions with the last approximately 9 amino acids in the conserved SSB C-terminal tails (SSB-Ct). We observe intrinsic specificity for the binding of an isolated SSB-Ct peptide to PriA over chi due primarily to a more favorable enthalpic component. PriA and chi also bind with weaker affinity to SSB (in the absence of ssDNA) than to isolated SSB-Ct peptides, indicating an inhibitory effect of the SSB protein core. Although the binding affinity of SSB for both chi and PriA is enhanced if SSB is prebound to ssDNA, this effect is larger with PriA indicating a further enhancement of SSB specificity for PriA. These results also suggest that DNA binding proteins such as PriA, which also interact with SSB, could use this interaction to gain access to ssDNA by first interacting with the SSB C-termini.

Human DNA Polymerase β Recognizes Single-stranded DNA Using Two Different Binding Modes

Interactions between the human DNA polymerase β (pol β) and a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence increase of the poly(dA) etheno-derivative (poly(dεA)) as a function of the binding density of pol β-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol β forms a complex with a ssDNA in which 16 nucleotides are occluded by the enzyme. In the second phase, transition to a complex where the polymerase occludes only 5 nucleotides occurs. Thus, human pol β binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol β)16 and the second as (pol β)5binding modes. The analyses of the enzyme binding to ssDNA have been performed using statistical thermodynamic models, which account for the existence of the two binding modes of the enzyme, cooperative interactions, and the overlap of potential binding sites. The importance of the discovery that human pol β binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol β, is discussed.

Quantitative analysis of ligand-macromolecule interactions using differential dynamic quenching of the ligand fluorescence to monitor the binding

Quantitative analyses of the thermodynamics and kinetics of ligand-macromolecule interactions in biological systems rely predominately on monitoring changes in the spectroscopic properties of the ligand or macromolecule, particularly fluorescence changes, which accompany the formation of the studied complexes. However, in many instances the interactions do not affect the fluorescence properties of interacting species and do not provide a resolution high enough to perform quantitative and rigorous measurements of the thermodynamic and/or kinetic parameters. In this communication, we describe the theoretical and experimental aspects of a method of studying complex, multiple ligand-macromolecule interactions by the fluorescence titration technique, when the intrinsic fluorescence changes accompanying binding do not provide a resolution necessary to perform quantitative analyses. The method is based on the fact that a fluorescent ligand, or binding sites of the macromolecule, can have different accessibility to the collisional (dynamic) quencher, when involved in the complex, rather than in the free, unbound state. The presence of an external dynamic quencher in solution, i.e., the presence of an extra collisional quenching process, transforms the fluorescence of the ligand or macromolecule, intrinsically independent of the complex formation, into a property which is dramatically different in the free state than in the bound state of the fluorophore. The approach is applicable to any model of noncooperative or cooperative ligand binding to a macromolecule and allows for the optimization of the resolution of the binding or kinetic studies for a given ligand-macromolecule system. The application of the method is illustrated by applying it to the study of the binding of the fluorescent derivative of a nucleotide cofactor, epsilon ADP, to the six interacting sites of the E. coli primary replicative helicase DnaB protein hexamer.

Interactions of the Escherichia coli primosomal PriB protein with the single-stranded DNA. Stoichiometries, intrinsic affinities, cooperativities, and base specificities.

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12+/-1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.

Kinetics of allosteric conformational transition of a macromolecule prior to ligand binding: analysis of stopped-flow kinetic experiments.

Two fundamentally different mechanisms of ligand binding are commonly encountered in biological kinetics. One mechanism is a sequential multistep reaction in which the bimolecular binding step is followed by first-order steps. The other mechanism includes the conformational transition of the macromolecule, before the ligand binding, followed, by the ligand binding process to one of the conformational states. In stopped-flow kinetic studies, the reaction mechanism is established by examining the behavior of relaxation times and amplitudes as a function of the reactant concentrations. A major diagnostic tool for detecting the presence of a conformational equilibrium of the macromolecule, before the ligand binding, is the decreasing value of one of the reciprocal relaxation times with the increasing [ligand]. The sequential mechanism cannot generate this behavior for any of the relaxation times. Such dependence is intuitively understood on the basis of approximate expressions for the relaxation times that can be comprehensively derived, using the characteristic equation of the coefficient matrix and polynomial theory. Generally, however, the used approximations may not be fulfilled. On the other hand, the two kinetic mechanisms can always be distinguished, using the approach based on the combined application of pseudo-first-order conditions, with respect to the ligand and the macromolecule. The two experimental conditions differ profoundly in the extent of the effect of the ligand on the protein conformational equilibrium. In a large excess of the ligand, the conformational equilibrium of the macromolecule, before the ligand binding, is strongly affected by the binding process. However, in a large excess of the macromolecule, ligand binding does not perturb the internal equilibrium of the macromolecule. As a result, the normal mode, affected by the conformational transition, is absent in the observed relaxation process. In the case of a sequential mechanism, the number of relaxation times is not altered by different pseudo-first-order conditions. Thus, the approach provides a strong diagnostic criterion for detecting the presence of the conformational transition of the macromolecule and establishing the correct mechanism. Application of this approach is illustrated for the binding of 3′-O-(N-methylantraniloyl)-5′-diphosphate to the E. coli DnaC protein.

Dynamics of gapped DNA recognition by human polymerase β

Kinetics of human polymerase β binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ∼600–1200 s−1. This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO 4 − group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap.