Biomedical subjects

W Bujalowski

Publications and source records attributed to W Bujalowski.

At least 37 records · Page 2Linked to original sources

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↗

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↗

PubMed1992

Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions.

The Escherichia coli single-strand binding (SSB) protein is an essential protein involved in DNA replication, recombination, and repair processes. The tetrameric protein binds to ss nucleic acids in a number of different binding modes in vitro. These modes differ in the number of nucleotides occluded per SSB tetramer and in the type and degree of cooperative complexes that are formed with ss DNA. Although it is not yet known whether only one or all of these modes function in vivo, based on the dramatically different properties of the SSB tetramer in these different ss DNA binding modes, it has been suggested that the different modes may function selectively in replication, recombination, and/or repair. The transitions between these different modes are very sensitive to solution conditions, including salt (concentration, as well as cation and anion type), pH, and temperature. We have examined the effects of multivalent cations, principally the polyamine spermine, on the SSB-ss poly(dT) binding mode transitions and find that the transition from the (SSB)35 to the (SSB)56 binding mode can be induced by micromolar concentrations of polyamines as well as the inorganic cation Co(NH3)6(3+). Furthermore, these multivalent cations, as well as Mg2+, induce the binding mode transition by binding cooperatively to the SSB-poly(dT) complexes. These observations are interesting in light of the fact that polyamines, such as spermidine, are part of the ionic environment in E. coli and hence these cations are likely to affect the distribution of SSB-ss DNA binding modes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗

PubMed1992

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗

PubMed1991

Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein.

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

DNA-Binding Proteins↗

PubMed1991

Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA.

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase. However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.

Bacterial Proteins↗

PubMed1991

Thermodynamic methods for model-independent determination of equilibrium binding isotherms for protein-DNA interactions: spectroscopic approaches to monitor binding.

The measurement of equilibrium binding constants for ligand-macromolecule interactions by monitoring a change in some spectral property of the ligand or the macromolecule is a common method used to study these interactions. This is due to the high sensitivity of the spectroscopic methods and general ease in applying these experimental procedures. In addition, binding can be monitored continuously, thus facilitating kinetic measurements. The main problem with these methods results from the fact that the spectroscopic signal is an indirect measure of binding, since the relationship between the change in the spectroscopic signal and the extent of binding is unknown, a priori. A common recourse is to assume a strict proportionality between the signal change and the fractional saturation of the ligand or macromolecule; however, it is often the case that such a direct proportionality does not hold. In this chapter we have reviewed the use of methods to analyze ligand-macromolecule equilibrium titrations that are monitored by indirect spectroscopic techniques. These methods of analysis yield thermodynamically rigorous, model-independent binding isotherms, hence assumptions concerning the relationship between the signal change and the extent of binding are not required. In fact, these methods can also be used to determine quantitatively the relationship between the signal change and the average degree of binding. In addition, the approaches discussed here are general and not limited to spectroscopic signals and therefore can be used with any intensive physicochemical property that reflects binding.

DNA↗

PubMed1989

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of DNA is highly specific for duplex DNA.

We show that iron(II)-EDTA-catalyzed cleavage of duplex DNA is much more rapid than cleavage of single-stranded DNA that does not form intramolecular base pairs. Comparisons of the extent of cleavage of the fully single-stranded oligonucleotides d(pT)70, d(pA)70, and d(pC)35 and the duplex DNA d(pT)70.d(pA)70 indicate that the extent of cleavage increases significantly upon formation of the duplex structure. These observations indicate that accessibility of the DNA sugars to the presumed cleaving agent, hydroxyl radical, is not the major determinant for cleavage and that most likely a direct interaction between Fe(II) and the DNA is required. As a result, the interpretation of DNA cleavage experiments performed with this reagent to obtain detailed structural information should be pursued with caution until the mechanism of the cleavage reaction is better understood.

DNA↗