Biomedical subjects

W Bujalowski

Publications and source records attributed to W Bujalowski.

At least 19 recordsLinked to original sources

PubMed1998

Transition between different binding modes in rat DNA polymerase beta-ssDNA complexes.

Interactions of rat DNA polymerase beta with a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence changes accompanying the binding, as a function of the thermodynamically rigorous binding density of rat pol beta-ssDNA complexes, reveals the existence of two binding phases. In the first high affinity phase, rat pol beta forms a complex with the ssDNA in which 16 nucleotides are occluded by the enzyme. In the second low affinity phase, a transition to a complex where the polymerase occludes only five nucleotides occurs. Thus, the data show that rat pol beta binds the ssDNA in two binding modes which differ in the number of occluded nucleotides. We designate the first complex as the (pol beta)16 binding mode and the second as the (pol beta)5 binding mode. The formation of the (pol beta)16 and (pol beta)5 modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol beta)16 or the (pol beta)5 mode, and a 10mer which can form only the (pol beta)5 mode. Binding of rat pol beta to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding modes, cooperative interactions, and the overlap of potential binding sites. The results indicate that the 8 kDa domain of the enzyme is involved in ssDNA binding in both modes. Binding studies show that an isolated 8 kDa domain has the same intrinsic affinity for the ssDNA as the entire intact enzyme in its (pol beta)5 mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol beta)5 mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions released, and a lower fluorescence change accompanying the formation of the (pol beta)16 than the (pol beta)5 mode indicate that the 31 kDa catalytic domain of the enzyme interacts with the ssDNA only in the (pol beta)16 mode. The significance of these results for understanding the functioning of rat pol beta in the DNA metabolism is discussed.

Animals↗

PubMed1998

Human DNA polymerase beta recognizes single-stranded DNA using two different binding modes.

Interactions between the human DNA polymerase beta (pol beta) 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(depsilonA)) as a function of the binding density of pol beta-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol beta 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 beta binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol beta)16 and the second as (pol beta)5 binding 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 beta binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol beta, is discussed.

Allosteric Regulation↗

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↗

PubMed1998

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 approximately 10 nucleotides. ssDNA affinity for the strong subsite is approximately 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.

Bacterial Proteins↗

PubMed1998

Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure.

Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.

Adenylyl Imidodiphosphate↗

PubMed1997

Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork.

The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.

Bacterial Proteins↗

PubMed1997

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.

Adenosine Diphosphate↗

PubMed1996

The hexameric E. coli DnaB helicase can exist in different Quaternary states.

The DnaB protein is the primary replicative helicase in Escherichia coli, and the active form of the protein is a hexamer. It has been reported that the protein forms a ring with strong 3-fold symmetry, which was suggested to be a trimer of dimers. We show that under different conditions, using either ATP, ATP gamma S, AMP-PNP or ADP as nucleotide cofactors, we always find two different forms of the DnaB ring; one with a 3-fold symmetry and one with 6-fold symmetry. We have used scanning transmission electron microscopy for mass analysis, and have found that both forms are hexamers, excluding the possibility that the 3-fold form is in fact a trimer of the 52 kDa monomer. We have also found rings that are in an intermediate state between these two. The existence of hexamers in discrete states shows that the transitions between these states must be cooperative. These observations suggest that there may be an equilibrium between two different conformations of the hexameric ring. The role of these two states in the mechanism of helicase action remains to be determined.

Adenosine Diphosphate↗

PubMed1996

Global conformational transitions in Escherichia coli primary replicative helicase DnaB protein induced by ATP, ADP, and single-stranded DNA binding. Multiple conformational states of the helicase hexamer.

The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is s20,w = 11.9 +/- 0.2 compared to the sedimentation coefficient s20,w = 10.5 +/- 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 +/- 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-depsilonA(pepsilonA)19, s20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a "closed" conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a "tense" state which binds ssDNA with an affinity of approximately 4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a "relaxed" conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant approximately 3 orders of magnitude weaker than in the presence of the ATP analog (tense state).

Adenosine Diphosphate↗

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↗

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↗

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↗

PubMed1995

Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA. The nucleic acid does not wrap around the protein hexamer.

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)

Bacterial Proteins↗

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↗

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↗

PubMed1994

Effects of base composition on the negative cooperativity and binding mode transitions of Escherichia coli SSB-single-stranded DNA complexes.

We have examined the ability of the Escherichia coli single-stranded DNA binding protein (SSB) tetramer to form its different binding modes on poly(dC), poly(U), and poly(A) over a range of NaCl and NaF concentrations for comparison with previous studies with poly(dT). In reverse titrations with poly(U) and poly(A) at 25 degrees C, pH 8.1, SSB forms all four binding modes previously observed with poly(dT), namely, (SSB)35, (SSB)40, (SSB)56, and (SSB)65, where the subscript denotes the site size (i.e., the average number of nucleotides occluded per SSB tetramer). As with poly(dT), the low site size modes are favored at low monovalent salt concentration (< 10 mM), whereas increasing salt concentration facilitates the transitions to the higher site size modes. Surprisingly, SSB does not form a stable (SSB)35 complex on poly(dC), even at 1 mM NaCl; rather, the (SSB)56 mode is formed under these conditions. Upon raising the [NaCl], the (SSB)56 complex undergoes a transition to the (SSB)65 complex (transition midpoint, 40 mM NaCl). On the basis of studies with dC(pC)34, dT(pT)34, and dA(pA)34, the inability of the SSB tetramer to form the (SSB)35 complex with poly(dC) is due mainly to a much lower degree of negative cooperativity for binding oligodeoxycytidylates to the SSB tetramer. At low salt concentration, the negative cooperativity parameter, sigma 35, is lowest for dA(pA)34, intermediate for dT(pT)34, and highest for dC(pC)34, indicating that it is most difficult to saturate the SSB tetramer with two molecules of dA(pA)34. We have also measured the equilibrium constants for binding the oligodeoxynucleotides dC(pC)34, dC(pC)69, dA(pA)34, and dA(pA)69 as a function of [NaCl] and [NaBr] and find that the salt dependencies of these oligonucleotides are dependent upon base composition. These studies also indicate that ion binding accompanies formation of these SSB-ss-DNA complexes, although there is a net release of ions upon formation of the complex. This influence of both salt concentration and base composition indicates that both electrostatic and nonelectrostatic factors contribute to the negative cooperativity associated with ss-DNA binding to the SSB tetramer.

Base Composition↗

PubMed1994

Structural characteristics of the nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein. Studies with ribose and base-modified fluorescent nucleotide analogs.

Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (epsilon ADP) and the ribose-modified fluorescent analogs 3'(2')-O-(N-methylantraniloyl)adenosine 5'-diphosphate (MANT-ADP), 3'-O-(N-methylantraniloyl)deoxyadenosine 5'-diphosphate (MANT-dADP), 3'-O-(N-methylantraniloyl)-deoxyadenosine 5'-triphosphate (MANT-dATP), and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of epsilon ADP to the DnaB helicase causes only approximately 21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound epsilon ADP is characterized by a single lifetime of 24.2 +/- 0.6 ns, only slightly shorter than the fluorescent lifetime of the free epsilon ADP in solution (25.5 +/- 0.6 ns). Solute-quenching studies of bound epsilon ADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound epsilon ADP is 0.21 +/- 0.02, compared to the anisotropy of 0.3 of completely immobilized epsilon ADP at the same excitation wavelength (lambda ex = 325 nm, lambda em = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2' and/or 3' oxygens of the ribose, increases upon binding to DnaB by factors of approximately 4.7 (lambda ex = 408 nm) and approximately 2.6 (lambda ex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by approximately 11 nm and that of MANT-ADP by approximately 12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine↗

PubMed1994

Co-operative binding of Escherichia coli SSB tetramers to single-stranded DNA in the (SSB)35 binding mode.

Escherichia coli SSB tetramers can bind to single stranded (ss) DNA in several binding modes. At 25 degrees C, pH 8.1, SSB can form at least three distinct binding modes, (SSB)n, where the number of nucleotides occluded per tetramer (n), can have values of 35, 56 or 65. Stability of the different modes is modulated by solution conditions, primarily the salt concentration and type, as well as the free SSB concentration. At least two different types of positive co-operative binding of SSB to ssDNA have also been observed, which appear to be correlated with different SSB binding modes. The (SSB)65 mode, which dominates at monovalent salt concentrations > 0.2 M, displays only moderate, "limited" co-operative binding in which clustering of SSB is limited to the formation of dimers of tetramers (octamers). However, at lower salt concentrations, "unlimited" co-operative binding is observed in which long SSB clusters can form, similar to the behavior observed for the phage T4 gene 32 protein. It has been proposed that unlimited co-operativity is linked to the (SSB)35 binding mode; however, this has not been verified since quantitative estimates of the co-operativity in this binding mode are difficult on long ssDNA. To estimate the nearest-neighbor co-operativity parameter in the (SSB)35 mode, we have examined the equilibrium binding of SSB to the oligodeoxynucleotide, dA(pA)69. Under certain conditions, 1:1 complexes, in which all four SSB subunits interact with the dA(pA)69, can form at low SSB binding densities, whereas 2:1 complexes, in which both SSB tetramers bind to DNA using only two subunits, can form at high SSB binding densities. These 2:1 complexes serve as a model for co-operative binding in the (SSB)35 binding mode. We show that SSB tetramers bind in this mode with a minimum nearest-neighbor co-operativity parameter of omega 35 = 1.0 x 10(5) (0.125 M NaCl, pH 8.1, 25 degrees C). This indicates that the nearest-neighbor co-operativities for SSB tetramers bound to ssDNA in the (SSB)35 versus the (SSB)65 mode differ qualitatively and quantitatively and suggests that the (SSB)35 mode is responsible for the ability of SSB protein to form long clusters on ssDNA. If the ability of helix destabilizing proteins to form uninterrupted protein clusters on ssDNA is important in DNA replication, then it is likely that SSB uses its (SSB)35 mode to function in this capacity.

Binding Sites↗