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PubMed2000

pH-controlled quaternary states of hexameric DnaB helicase.

DnaB is the major helicase in the Escherichia coli replisome. It is a homohexameric enzyme that interacts with many other replisomal proteins and cofactors. It is usually loaded onto a single strand of DNA at origins of replication from its complex with its loading partner DnaC, then translocates in the 5' to 3' direction, unwinding duplex DNA in an NTP-driven process. Quaternary polymorphism has been described for the DnaB oligomer, a feature it has in common with some other hexameric helicases. In the present work, electron microscopy and in- depth rotational analysis studies of negatively stained specimens has allowed the establishment of conditions that govern the transition between the two different rotational symmetry states (C(3) and C(6)) of DnaB. It is shown: (a) that the pH value of the sample buffer, within the physiological range, dictates the quaternary organisation of the DnaB oligomer; (b) that the pH-induced transition is fully reversible; (c) that the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB.DnaC complex exists only as particles with C(3) symmetry; and (e) that DnaC interacts only with DnaB particles that have C(3) symmetry. Structural consequences of this quaternary polymorphism, as well as its functional implications for helicase activity, are discussed.

Adenosine Diphosphate↗

PubMed2006

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex.

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.

Adenosine Diphosphate↗

PubMed2000

tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain IV, located within the unique C terminus of tau, binds the replication fork, helicase, DnaB.

Interaction between the tau subunit of the DNA polymerase III holoenzyme and the DnaB helicase is critical for coupling the replicase and the primosomal apparatus at the replication fork (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In the preceding manuscript, we reported the identification of five putative structural domains within the tau subunit (Gao, D., and McHenry, C. (2000) J. Biol. Chem. 275, 4433-4440). As part of our systematic effort to assign functions to each of these domains, we expressed a series of truncated, biotin-tagged tau fusion proteins and determined their ability to bind DnaB by surface plasmon resonance on streptavidin-coated surfaces. Only tau fusion proteins containing domain IV bound DnaB. The DnaB-binding region was further limited to a highly basic 66-amino acid residue stretch within domain IV. Unlike the binding of immobilized tau(4) to the DnaB hexamer, the binding of monomeric domain IV to DnaB(6) was dependent upon the density of immobilized domain IV, indicating that DnaB(6) is bound by more than one tau protomer. This observation implies that both the leading and lagging strand polymerases are tethered to the DnaB helicase via dimeric tau. These double tethers of the leading and lagging strand polymerases proceeding through the tau-tau link and an additional tau-DnaB link are likely important for the dynamic activities of the replication fork.

Bacterial Proteins↗

PubMed1999

Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101.

Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA-DnaB interaction cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo. Although, interaction of DnaA with DnaB could not substitute for RepA-DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.

Bacterial Proteins↗

PubMed1996

A general method of analysis of ligand binding to competing macromolecules using the spectroscopic signal originating from a reference macromolecule. Application to Escherichia coli replicative helicase DnaB protein nucleic acid interactions.

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.

Bacterial Proteins↗

PubMed2002

Escherichia coli DnaA protein loads a single DnaB helicase at a DnaA box hairpin.

The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure.

Bacterial Proteins↗

PubMed2000

The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase.

Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.

Amino Acid Sequence↗

PubMed1998

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 the Escherichia 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 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) 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.

Bacterial Proteins↗

PubMed1994

Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.

Bacterial Proteins↗

PubMed1996

Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA.

The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins. In this paper we present evidence supporting direct interaction between the two proteins. We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interation also cause a significant reduction in primer synthesis. Thus, the physical interaction reported here appears to be physiologically significant.

Amino Acid Sequence↗

PubMed1994

Oligomeric structure of Escherichia coli primary replicative helicase DnaB protein.

The oligomeric structure of the Escherichia coli primary replicative helicase DnaB protein in relation to the functions of the enzyme and the energetics of its stability has been characterized. Sedimentation equilibrium, sedimentation velocity, and ligand binding studies show that, in solutions containing magnesium ions, the DnaB helicase exists as a stable hexamer over a wide protein concentration range (approximately 10(-7) to 10(-5) M (hexamer)). The sedimentation coefficient of the hexamer (s0(20,w) = 10.3 +/- 0.3 S) provides an apparent frictional ratio of 1.09 +/- 0.03, which suggests that the hexamer has a nonspherical shape and, when modeled as a prolate ellipsoid of revolution, has an axial ratio of a/b = 2.6 +/- 0.6. Magnesium ions play a crucial structural role in stabilizing the hexameric structure of the DnaB helicase. In the absence of Mg2+, the DnaB protein forms a trimer that, at low protein concentrations, dissociates into monomers. Analysis of the sedimentation data indicates that the dimerization of the trimers into the active DnaB hexamer is accompanied by an uptake of approximately 4 magnesium cations. The sedimentation coefficient of the DnaB monomer (s0(20,w) = 2.8 +/- 0.3 S) provides an apparent frictional ratio of 1.22 +/- 0.05, which indicates that the monomer has an elongated structure with an axial ratio of a/b = 5.2 +/- 0.8 when modeled as a prolate ellipsoid of revolution. Analysis of the ratio of the sedimentation coefficients (the sedimentation ratio) of the DnaB hexamer and monomer, which depends solely on the shape of the protomer and the mode of aggregation, strongly suggests that elongated DnaB promoters aggregate with cyclic symmetry in which the protomer-protomer contacts are limited to only two neighboring subunits.

Adenine Nucleotides↗

PubMed1993

Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs.

The interactions of nucleotides with Escherichia coli replicative helicase DnaB protein have been systematically studied using fluorescent nucleotide analogs, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), 3'-O-(N-methylantraniloyl) 5'-diphosphate (MANT-ADP), and 1,N6-ethenoadenosine diphosphate (epsilon ADP). The binding of the analogs is accompanied by strong quenching of the protein fluorescence; 0.76 +/- 0.05, 0.76 +/- 0.05, 0.58 +/- 0.05, and 0.53 +/- 0.5 for TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, respectively. A thermodynamically rigorous method has been applied to obtain all binding parameters from fluorescence titration curves independent of the assumption of strict proportionality between the observed quenching of the protein fluorescence and the degree of nucleotide binding. An exact representation of the observed fluorescence quenching, as a function of the nucleotide binding, is introduced through an empirical function which enables analysis of single binding isotherms without the necessity of determining all quenching constants for different binding sites. Using this method, we determined that, at saturation, the DnaB hexamer binds six molecules of TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, and that there is strong heterogeneity among nucleotide binding sites. The binding isotherms are biphasic. Three molecules of nucleotide are bound in the first high-affinity binding phase, and the subsequent three molecules are bound in the second low-affinity binding phase. The separation of the two binding steps is even more pronounced at higher temperatures. The change of the monitored fluorescence is sequential. The binding of the first nucleotide causes the largest quenching of the protein fluorescence with subsequent nucleotide binding inducing progressively less quenching. The simplest explanation of this behavior is that there is a negative cooperativity among nucleotide binding sites on a DnaB hexamer. The negative cooperativity is an intrinsic property of the DnaB helicase, since it is observed in the binding of nucleotide analogs which are different in type and location of the modifying group. A statistical thermodynamic model is proposed, the hexagon, which provides an excellent description of the binding process using only two interaction parameters, intrinsic binding constant K and cooperativity parameter sigma. The data suggest an important role of the phosphate groups in binding and in recognition of nucleotides by the DnaB helicase.

Adenosine↗

PubMed1996

Binding of Escherichia coli primary replicative helicase DnaB protein to single-stranded DNA. Long-range allosteric conformational changes within the protein hexamer.

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 = 12.3 +/- 0.3, compared with S20,W = 10.5 +/- 0.3 of the free enzyme, indicating large changes in the hydrodynamic properties of the enzyme in the complex. These results provide direct evidence that the DnaB hexamer undergoes dramatic conformational changes which include all six subunits of the enzyme in the ternary complex. Moreover, sedimentation velocity studies of the ternary complex provide direct evidence that the hexamer is the species which binds ss nucleic acid. The significance of these results for a mechanistic model of the functioning of the DnaB helicase in DNA replication is discussed.

Allosteric Regulation↗

PubMed2000

Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues.

The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues bearing the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, epsilonAMP-PNP and epsilonADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four-steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: [equation in text]. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)(1). Moreover, the value of the bimolecular rate constant, k(1), is 3-4 orders of magnitude lower than the value expected for the diffusion-controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.

Adenosine Diphosphate↗

PubMed1996

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 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.

Adenosine Diphosphate↗

PubMed2000

DnaB helicase stimulates primer synthesis activity on short oligonucleotide templates.

DnaB helicase stimulated the second-order RNA primer synthesis activity of primase by over 5000-fold on DNA templates that were 23 nucleotides long. This template length is the same as the DnaB helicase thermodynamic binding site size [Jezewska, M. J., and Bujalowski, W. (1996) Biochemistry 35, 2117-2128]. This phenomenal stimulation was achieved by increasing the template affinity of primase by over 300-fold and increasing the catalytic rate by over 15-fold. It was necessary to determine the optimal amount of DnaB helicase to achieve this stimulation because helicase stimulation was cooperative at low concentration and inhibitory at high helicase concentration. The cooperative stimulation at low concentration indicated the presence of a time-dependent assembly step that preceded the active state. Besides stimulating primase activity, DnaB helicase also prevented primase from synthesizing RNA primers that were longer than the template sequence. In the absence of DnaB helicase, the majority of primers synthesized by primase were longer than the template and were named "overlong primers" [Swart, J. R., and Griep, M. A. (1995) Biochemistry 34, 16097-16106]. In contrast, the helicase-stimulated RNA primers were from 10 to 14 nucleotides in length with the 12-mer representing the majority of the total RNA primers produced. It was shown that DnaB helicase stabilized the open or single-stranded conformation of the template, which favored the synthesis of the template-length-dependent primers. In contrast, when primase acted alone, it stabilized the 3'-end hairpin conformation of the template so that the template's 3'-hydroxyl served as a "DNA primer" from which primase elongated to create the overlong primers.

Adenosine Triphosphate↗

PubMed1999

NMR structure of the N-terminal domain of E. coli DnaB helicase: implications for structure rearrangements in the helicase hexamer.

BACKGROUND: DnaB is the primary replicative helicase in Escherichia coli. Native DnaB is a hexamer of identical subunits, each consisting of a larger C-terminal domain and a smaller N-terminal domain. Electron-microscopy data show hexamers with C6 or C3 symmetry, indicating large domain movements and reversible pairwise association. RESULTS: The three-dimensional structure of the N-terminal domain of E. coli DnaB was determined by nuclear magnetic resonance (NMR) spectroscopy. Structural similarity was found with the primary dimerisation domain of a topoisomerase, the gyrase A subunit from E. coli. A monomer-dimer equilibrium was observed for the isolated N-terminal domain of DnaB. A dimer model with C2 symmetry was derived from intermolecular nuclear Overhauser effects, which is consistent with all available NMR data. CONCLUSIONS: The monomer-dimer equilibrium observed for the N-terminal domain of DnaB is likely to be of functional significance for helicase activity, by participating in the switch between C6 and C3 symmetry of the helicase hexamer.

Amino Acid Sequence↗

PubMed1994

Close proximity of tryptophan residues and ATP-binding site in Escherichia coli primary replicative helicase DnaB protein. Molecular topography of the enzyme.

The binding of fluorescent nucleotide analogs to the Escherichia coli primary replicative helicase DnaB protein causes strong quenching of protein tryptophan fluorescence. It results from the efficient fluorescence energy transfer (E) from tryptophans to analogs bound in the nucleotide-binding site, indicating that protein tryptophans are "clustered" in close proximity to the binding site. This is in contrast to the lack of detectable energy transfer to the fluorescent single-stranded DNA (ssDNA) derivative, suggesting a distant separation between two function-linked structural elements of the enzyme, the nucleotide- and ssDNA-binding sites. The dependence of E upon the average number of bound nucleotides/DnaB hexamer is nonlinear, implying a larger separation between tryptophans and the bound nucleotide in the low affinity sites. Spectroscopic studies reveal that tryptophan residues are located on the surface of the DnaB helicase in a hydrophobic cleft, whereas the environment of the tyrosines is heterogeneous, with 6 out of 10 tyrosine residues located on the surface of the helicase. The efficiency of the fluorescence energy transfer from the tyrosines to tryptophans suggests that the "centers of mass" of the residues are separated, possibly reflecting the separation of the nucleotide- and ssDNA-binding sites, with tyrosines constituting part of the ssDNA-binding region.

Acrylamide↗