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Wlodzimierz Bujalowski

Publications and source records attributed to Wlodzimierz Bujalowski.

At least 19 recordsLinked to original sources
PubMed2006

The Escherichia coli PriA helicase has two nucleotide-binding sites differing dramatically in their affinities for nucleotide cofactors. 1. Intrinsic affinities, cooperativities, and base specificity of nucleotide cofactor binding.

Interactions of the Escherichia coli PriA helicase with nucleotide cofactors have been studied using the fluorescence titration and analytical ultracentrifugation techniques. Binding of unmodified cofactors was characterized by the fluorescence competition titration method. The obtained data establish that at saturation the PriA helicase binds two nucleotide molecules per protein monomer. This result corroborates with the primary structure of the protein, which contains sequence motifs implicated as putative nucleotide-binding sites. The intrinsic affinities of the binding sites differ by 2-4 orders of magnitude. Thus, the PriA helicase has a strong and a weak nucleotide-binding site. The binding sites differ dramatically in their properties. The strong site is highly specific for adenosine cofactors, while the weak site shows very modest base specificity. The affinities of the strong and weak binding sites for ATP are lower than the affinities for ADP, although both sites have similar affinity for the inorganic phosphate group. Unlike the weak site, the affinity of the strong site profoundly depends on the structure of the phosphate group of the ATP cofactor. Binding of unmodified nucleotides indicates the presence of positive cooperative interactions between bound cofactors (i.e., the existence of communication between the two sites). Magnesium cations are specifically involved in controlling the cofactor affinity for the strong site, while the affinity of the weak site is predominantly determined by interactions between the phosphate group and ribose regions of the cofactor and the protein matrix. The significance of these results for the activities of the PriA helicase is discussed.

Adenosine Diphosphate↗
PubMed2006

Kinetic mechanisms of the nucleotide cofactor binding to the strong and weak nucleotide-binding site of the Escherichia coli PriA helicase. 2.

Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.

Adenosine Diphosphate↗
PubMed2006

Allosteric interactions between the nucleotide-binding sites and the ssDNA-binding site in the PriA helicase-ssDNA complex. 3.

Allosteric interactions between the strong and weak nucleotide-binding sites and the total and proper single-stranded (ss)DNA-binding sites of the Escherichia coli PriA helicase have been analyzed using the fluorescence titration technique. Binding of the DNA exclusively to the proper DNA-binding site of the helicase, profoundly affects the intrinsic affinities of both nucleotide-binding sites, indicating a direct communication between the nucleotide-binding sites and the proper DNA-binding site. The communication involves conformational changes of the entire protein molecule. Nevertheless, the bound DNA differently affects the structures of the strong and weak nucleotide-binding sites. While the polarity of the strong site is moderately diminished, the polarity of the weak site is dramatically increased, indicating an intimate involvement of the weak site in controlling the helicase interactions with the DNA. The strong site does not directly control the DNA affinity of the enzyme. Only when the helicase has both nucleotide-binding sites saturated with ADP but not with ATP analogues does the enzyme have an increased affinity for the ssDNA, indicating that the control of ssDNA affinity involves a coordinated action of both nucleotide-binding sites and depends upon the phosphate group of the bound cofactor. A dramatic increase of the DNA affinity, when the DNA encompasses the total DNA-binding site of the enzyme, with both nucleotide-binding sites saturated with ADP or NDP, indicates that an additional area of the protein within the total DNA-binding site becomes engaged in interactions with the DNA. The significance of these results for the enzyme activities in the DNA unwinding and recognition is discussed.

Adenosine Diphosphate↗
PubMed2005

DNA polymerase X from African swine fever virus: quantitative analysis of the enzyme-ssDNA interactions and the functional structure of the complex.

Interactions of polymerase X from African swine fever virus with single-stranded DNA (ssDNA) have been studied, using quantitative fluorescence titration and analytical ultracentrifugation techniques. Experiments were performed with a fluorescent etheno-derivative of ssDNA oligomers. Studies of unmodified ssDNA oligomers were carried out using the competition titration method. The total site-size of the pol X-ssDNA complex is 16(+/-1) nucleotide residues. The large total ssDNA-binding site has a complex heterogeneous structure. It contains the proper ssDNA-binding site that encompasses only 7(+/-1) residues. As the length of the ssDNA increases, the enzyme engages an additional binding area in interactions with the DNA, at a distance of approximately 7-8 nucleotides from the proper site, which is located asymmetrically within the polymerase molecule. As a result, the net ion release accompanying the interactions with the DNA, increases from approximately 1 for the proper DNA-binding site to approximately 6 for the total DNA-binding site. Unlike in the case of the mammalian polymerase beta that belongs to the same polymerase X family, the DNA-binding areas within the total DNA-binding site of pol X are not autonomous. Consequently, the enzyme does not form different binding modes with different numbers of occluded nucleotide residues, although the interacting areas are structurally separated. The statistical thermodynamic model that accounts for the engagement of the proper and the total DNA-binding site in interactions with the DNA provides an excellent description of the binding process. Pol X binds the ssDNA without detectable cooperativity and with very modest base specificity.

African Swine Fever Virus↗
PubMed2005

Binding of six nucleotide cofactors to the hexameric helicase RepA protein of plasmid RSF1010. 2. Base specificity, nucleotide structure, magnesium, and salt effect on the cooperative binding of the cofactors.

Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of approximately 10 and approximately 1000 higher than the value observed for ADP and PO(4)(-). The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the gamma phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg(2+), to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg(2+) cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg(2+) has a dual function in nucleotide-helicase interactions. At low [Mg(2+)], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg(2+)], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base.

Adenosine Diphosphate↗
PubMed2005

Binding of six nucleotide cofactors to the hexameric helicase RepA protein of plasmid RSF1010. 1. Direct evidence of cooperative interactions between the nucleotide-binding sites of a hexameric helicase.

The interactions of nucleotides with RepA hexameric helicase from plasmid RSF1010 have been examined using nucleotide analogues, TNP-ADP, TNP-ATP, and MANT-ADP. The binding of the analogues is accompanied by strong quenching of the protein fluorescence. A quantitative fluorescence titration method has been applied to analyze the interactions, independent of any assumptions of proportionality between the fluorescence quenching and the average degree of binding. The fluorescence quenching as a function of the average degree of binding is expressed by an empirical function that enables analysis of the data, without the necessity of determining quenching parameters for different complexes. At saturation, the RepA hexamer binds six nucleotide molecules, indicating that each subunit of the hexamer can engage in interactions with the cofactor. The nucleotide macroscopic affinity decreases with the increasing degree of binding, indicating heterogeneity among the binding sites. A statistical thermodynamic hexagon model provides an excellent description of the binding process and requires only two interaction parameters, the intrinsic binding constant, K, and cooperativity parameter, sigma. The heterogeneity in affinity reflects negative cooperative interactions between the binding sites. Analyses of the data provide clear evidence that the alternative model of two independent classes of binding sites does not describe the nucleotide binding. Such a model cannot account for both, the binding isotherms and the dependence of the fluorescence quenching upon the degree of binding. Thus, cooperative interactions between the nucleotide-binding sites are an intrinsic property of the RepA helicase. The presence of the cooperative interactions indicates significant communication among the subunits of the helicase.

Adenosine Diphosphate↗
PubMed2005

Kinetic mechanism of rat polymerase beta-dsDNA interactions. Fluorescence stopped-flow analysis of the cooperative ligand binding to a two-site one-dimensional lattice.

Kinetics of cooperative binding of rat polymerase beta to a double-stranded DNA has been studied using the fluorescence stopped-flow techniques. The data have been analyzed by an approach developed to examine complete kinetics of cooperative large ligand binding to a one-dimensional lattice. The method is based on using the smallest possible system that preserves key ingredients of cooperative binding; i.e., at saturation, the lattice can accept only two ligand molecules. It allows the identification of collective amplitudes as well as amplitudes describing particular normal modes of the reaction. The mechanism of the intrinsic binding of pol beta to the dsDNA is different from the analogous mechanism for the ssDNA. The difference originates from different enzyme orientations in the corresponding complexes. Intrinsic binding to the dsDNA includes only two sequential steps: a very fast bimolecular association followed by an energetically favorable conformational transition of the complex. The transition following the bimolecular step does not facilitate the engagement of the enzyme in cooperative interactions. Its role seems to be reinforcing the affinity of the bimolecular step. Salt and magnesium cations affect both the bimolecular step and the conformational transition. As a result, the bimolecular step is less sensitive to the increased salt concentration, allowing the enzyme to preserve its initial dsDNA affinity. The changing character of cooperative interactions between bound enzyme molecules as a function of NaCl concentration and MgCl(2) concentration does not affect the binding mechanism. The engagement in cooperative interactions is approximately 3-4 orders of magnitude slower than the conformational transition of the DNA-bound polymerase. The importance of the obtained results for the pol beta activities is discussed.

Animals↗
PubMed2005

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

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

Adenosine Diphosphate↗
PubMed2005

The nucleotide-binding site of the Escherichia coli DnaC protein: molecular topography of DnaC protein-nucleotide cofactor complexes.

The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3'-O-(N-methylantraniloyl)-5'-triphosphate (MANT-ATP) and 3'-O-(N-methylantraniloyl)-5'-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.

Adenosine Diphosphate↗
PubMed2004

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method.

Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

Adenosine Triphosphate↗
PubMed2004

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: the effect of the 3' arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase.

The effect of two structural elements of a replication DNA fork substrate, the length of the 3' arm of the fork and the stability of the double-stranded DNA (dsDNA) part, on the kinetics of the dsDNA unwinding by the Escherichia coli hexameric helicase DnaB protein has been examined under single turnover conditions using the rapid quench-flow technique. The length of the 3' arm of the replication fork, i.e. the number of nucleotides in the arm, is a major structural factor that controls the unwinding rate and processivity of the helicase. The data show the existence of an optimal length of the 3' arm where there is the highest unwinding rate and processivity, indicating that during the unwinding process, the helicase transiently interacts with the 3' arm at a specific distance on the arm with respect to the duplex part of the DNA. Moreover, the area on the enzyme that engages in interactions has also a discrete size. For DNA substrates with the 3' arm containing 14, or less, nucleotide residues, the DnaB helicase becomes a completely distributive enzyme. However, the 3' arm is not a "specific activating cofactor" in the unwinding reaction. Rather, the 3' arm plays a role as a mechanical fulcrum for the enzyme, necessary to provide support for the advancing large helicase molecule on the opposite strand of the DNA. Binding of ATP is necessary to engage the 3' arm with the DnaB helicase, but it does not change the initial distribution of complexes of the enzyme with the DNA fork substrate. Stability of the dsDNA has a significant effect on the unwinding rate and processivity. The unwinding rate constant is a decreasing linear function of the fractional content of GC base-pairs in the dsDNA, indicating that the activation of the unwinding step is proportional to the stability of the nucleic acid.

Adenosine Triphosphate↗
PubMed2004

Interactions of the RepA helicase hexamer of plasmid RSF1010 with the ssDNA. Quantitative analysis of stoichiometries, intrinsic affinities, cooperativities, and heterogeneity of the total ssDNA-binding site.

Interactions between the replicative RepA helicase hexamer of plasmid RSF1010 with the single-stranded DNA (ssDNA) have been studied, using the quantitative fluorescence titration, analytical sedimentation velocity, and sedimentation equilibrium techniques. Experiments were performed with fluorescein-labeled ssDNA oligomers. Studies with unmodified ssDNA oligomers were accomplished using the macromolecular competition titration method. Analyses of RepA helicase interactions with a series of the ssDNA provide direct evidence that the total site-size of the RepA hexamer-ssDNA complex is 19 +/- 1 nucleotide residues. The total ssDNA-binding site of the hexamer has a heterogeneous structure. Part of the total binding site constitutes the proper ssDNA-binding site of the enzyme, an area that possesses strong ssDNA-binding capability and encompasses only 8 +/- 1 residues of the ssDNA. The statistical effect on the macroscopic binding constant for the proper ssDNA-binding site indicates that it is structurally separated from the remaining part of the total ssDNA-binding site. Engagement in interactions with the ssDNA is accompanied by net ion release. Moreover, the proper ssDNA-binding site shows little base specificity. On the other hand, with long ssDNA oligomers, the entire total ssDNA-binding site of the RepA hexamer engages in interactions with the ssDNA resulting in a dramatic change in the nature of interactions with the nucleic acid. The association includes an uptake of ions by the protein. Moreover, unlike the proper-ssDNA-binding site, the total binding site shows a significant preference for pyrimidine oligomers. In this aspect, the RepA helicase is different from the Escherichia coli DnaB hexamer that shows large preference for purine homo-oligomers. In similar solution conditions, the ssDNA intrinsic affinity of the RepA hexamer is similar to the intrinsic affinity of the DnaB helicase. The RepA helicase binds to ssDNA oligomers that can accept more than one RepA hexamer with significant positive cooperative interactions.

Bacterial Proteins↗
PubMed2004

Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors.

Global conformational and oligomeric states of the Escherichia coli replicative factor DnaC protein in the absence and presence of magnesium and nucleotide cofactors, ATP and ADP, and their fluorescent analogues, MANT-ATP and MANT-ADP, have been examined using analytical sedimentation velocity and time-dependent fluorescence anisotropy techniques. In solution, the DnaC protein exists exclusively as a monomer over a large protein concentration range. The value of s(degrees) (20, w)= 2.45 +/- 0.07 S indicates that the protein molecule has an elongated shape. When modeled as a prolate ellipsoid of revolution, the hydrated DnaC protein has an axial ratio of 4.0 +/- 0.6 with long axis a = 112 A and the short axis b = 28 A, respectively. The presence of magnesium or nucleotide cofactors, ATP or ADP, does not affect the global conformation of the protein and its monomeric state. These data indicate that recently found cooperative interactions between the DnaC molecules, in the complex with the DnaB helicase, are induced by the binding to the helicase, i.e., they are not the intrinsic property of the DnaC protein. Fluorescence anisotropy decays of the DnaC-MANT-ATP and DnaC-MANT-ADP complexes indicate that the protein has a rigid global structure on the nanosecond time scale, little affected by the nucleotide cofactors. Nevertheless, the complex with ATP has a more flexible structure, while the complex with ADP is more rigid, with the protein molecule assuming a more elongated shape. Magnesium exerts control only on the complex with the ATP analogue. In the absence of magnesium, the ATP analogue is firmly held in the binding site. In the presence of Mg(2+), this fixed location is released and the analogue is allowed to assume a flexible conformational state. The significance of the results for the functioning of the DnaC protein is discussed.

Adenosine Diphosphate↗
PubMed2004

Multistep sequential mechanism of Escherichia coli helicase PriA protein-ssDNA interactions. Kinetics and energetics of the active ssDNA-searching site of the enzyme.

Kinetics of the Escherichia coli PriA helicase interactions with the ssDNA has been studied, using the fluorescence stopped-flow technique. Experiments have been performed with a series of fluorescent etheno derivatives of ssDNA adenosine oligomers, differing in the number of nucleotide residues. The PriA helicase binds the ssDNA in the sequential process defined by [reaction: see text]. In the first step, the enzyme associates fast with the ssDNA without inducing conformational changes in the DNA. The dependence of the partial equilibrium constant, characterizing the first step, upon the length of the ssDNA strictly reflects the statistical relationship between the size of the DNA-binding site and the number of potential binding sites on the ssDNA. Only the DNA-binding site that encompasses 6.3 +/- 1 residues is directly involved in interactions. The site is located on a structural domain allowing the enzyme to efficiently search and recognize small patches of the ssDNA. Intramolecular steps are independent of the ssDNA length and accompanied by changes in the DNA structure. Salt and glycerol effects on the studied kinetics indicate a very different nature of the intermediates. While the bimolecular step is characterized by net ion release and water uptake, net ion uptake and water release accompany intramolecular transitions. Specific ion binding stabilizes the helicase-ssDNA complex in (P)(2) and (P)(3) intermediates. However, magnesium and AMP-PNP do not affect the mechanism of enzyme-ssDNA interactions. The sequential character of the mechanism indicates that the enzyme does not exist in a preequilibrium conformational transition prior to the DNA binding.

Adenosine Triphosphatases↗
PubMed2003

Tertiary conformation of the template-primer and gapped DNA substrates in complexes with rat polymerase beta. Fluorescence energy transfer studies using the multiple donor-acceptor approach.

The tertiary structure of template-primer and gapped DNA substrates in the complex with rat polymerase beta (pol beta) has been examined using the fluorescence energy transfer method based on the multiple donor-acceptor approach. In these studies, we used DNA substrates labeled at the 5' end of the template strand and the 5' end of the primer with the fluorescent donor and/or acceptor. Measurements of the enzyme complex with the template-primer DNA substrate having a ten nucleotide long ssDNA extension indicate that the distance between the 5' end of the template strand and the 5' end of the primer decreases by approximately 9.8 A as compared to the free nucleic acid. Analogous experiments with the template-primer substrate, having the ssDNA extension with five nucleotide residues, show approximately 6.6 A distance decrease. Such large distance decreases indicate that the DNA is significantly bent in the binding site. Analysis of the data indicates that the bending occurs between the third and the fourth nucleotide of the ssDNA extension. The entire template strand is at the bend angle Theta(TP) = 85 +/- 7 degrees with respect to the dsDNA part of the DNA molecule. In the polymerase complex with the gapped DNA, the distance between the 5' ends of the DNA and the bend angle are 66 +/- 2.2 A and 65 +/- 6 degrees, respectively. These values are very similar to the same distance and bend angle of the gap complex in the crystal structure of the co-complex. The presence of the 5'-terminal PO(4)(-) group downstream from the primer does not affect the tertiary conformation of the gapped DNA, indicating that the effect of the phosphate group is localized at the ssDNA gap.

Animals↗
PubMed2003

Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein. Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex.

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

Adenine Nucleotides↗
PubMed2003

Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries, intrinsic affinities, and cooperativities.

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.

Animals↗