Molecular analysis of barley stripe mosaic virus isolates differing in their biological properties and the development of reverse transcription loop-mediated isothermal amplification assays for their detection.

Barley stripe mosaic virus (BSMV) is an important seed-transmitted pathogen occurring worldwide. Recently, the occurrence of mild BSMV pathotypes has been observed in barley crops in Poland. In this study, the full-length genome sequences of mild and aggressive Polish and German BSMV isolates was established. Phylogenetic and recombination analysis was performed using Polish and other BSMV isolates described to date. The analysis revealed that Polish isolates differed only in 25 nucleotides, which suggests that point mutations might have had a great impact on the biological properties of the virus. The phylogenetic analysis revealed that the closest relationship was that between European and BSMV-CV42, BSMV-ND18 and BSMV-Type isolates, whereas the highest genetic distance was observed for BSMV-Qasr Ibrim and BSMV-China isolates. A recombination event within the αa protein of BSMV-De-M and BSMV-CV42 isolates was also detected. Moreover, a sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for rapid detection of BSMV isolates. The RT-LAMP assay can be used for routine diagnostics of BSMV in seed and plant material.

Stable Configurations of Charged Sedimenting Particles.

The qualitative behavior of charged particles in a vacuum is given by Earnshaw's theorem, which states that there is no steady configuration of charged particles in a vacuum that is asymptotically stable to perturbations. In a viscous fluid, examples of stationary configurations of sedimenting uncharged particles are known, but they are unstable or neutrally stable-they are not attractors. In this Letter, it is shown by example that two charged particles settling in a fluid may have a configuration that is asymptotically stable to perturbations for a wide range of charges, radii, and densities. The existence of such "bound states" is essential from a fundamental point of view and it can be significant for dilute charged particulate systems in various biological, medical, and industrial contexts.

Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings.

A one-step immunocapture real-time RT-PCR (IC-real-time RT-PCR) was developed for efficient detection of barley stripe mosaic virus (BSMV) in barley seedlings. The novel detection system was designed using a primer set targeting the conserved region in the triple gene block 2 (TGB2) to expand its capacity to detect all BSMV strains. This assay was evaluated for its efficiency in detecting BSMV in barley seedlings. Using the immunocapture sample preparation, real-time RT-PCR was able to detect BSMV in samples, which were indicated as negative by ELISA. The sensitivity of detection in the real-time RT-PCR was as low as 50 fg/µl of total viral RNA under optimal reaction conditions. This level of sensitivity indicated that the one-step IC-real-time RT-PCR developed in the present study could be used for routine plant and seed health assays.

Motion of a spherical particle near a planar fluid-fluid interface: the effect of surface incompressibility.

Hydrodynamic coupling of a spherical particle to an undeformable planar fluid-fluid interface under creeping-flow conditions is discussed. The interface can be either surfactant-free or covered with an incompressible surfactant monolayer. In the incompressible surfactant limit, a uniform surfactant concentration is maintained by Marangoni stresses associated with infinitesimal surfactant redistribution. Our detailed numerical calculations show that the effect of surface incompressibility on lateral particle motion is accurately accounted for by the first reflection of the flow from the interface. For small particle-interface distances, the remaining contributions are significant, but they are weakly affected by the surface incompressibility. We show that for small particle-wall gaps, the transverse and lateral particle resistance coefficients can be rescaled onto corresponding universal master curves. The scaling functions depend on a scaling variable that combines the particle-wall gap with the viscosity ratio between fluids on both sides of the interface. A logarithmic dependence of the contact value of the lateral resistance function on the viscosity ratio is derived. Accurate numerical calculations are performed using our Cartesian-representation method.

Hydrodynamic coupling of spherical particles to a planar fluid-fluid interface: theoretical analysis.

We have developed a new technique (based on our Cartesian-representation method) to describe hydrodynamic interactions of a spherical particle with an undeformable planar fluid-fluid interface under creeping-flow conditions. The interface can be either surfactant-free or covered with an incompressible surfactant monolayer. We consider the effect of surface incompressibility and surface viscosity on particle motion. The new algorithm allows to calculate particle mobility coefficients for hydrodynamically coupled particles, moving either on the same or on the opposite sides of the interface.

Streaming potential studies of colloid, polyelectrolyte and protein deposition.

Recent developments in the electrokinetic determination of particle, protein and polyelectrolyte monolayers at solid/electrolyte interfaces, are reviewed. Illustrative theoretical results characterizing particle transport to interfaces are presented, especially analytical formulae for the limiting flux under various deposition regimes and expressions for diffusion coefficients of various particle shapes. Then, blocking effects appearing for higher surface coverage of particles are characterized in terms of the random sequential adsorption model. These theoretical predictions are used for interpretation of experimental results obtained for colloid particles and proteins under convection and diffusion transport conditions. The kinetics of particle deposition and the structure of monolayers are analyzed quantitatively in terms of the generalized random sequential adsorption (RSA) model, considering the coupling of the bulk and surface transport steps. Experimental results are also discussed, showing the dependence of the jamming coverage of monolayers on the ionic strength of particle suspensions. In the next section, theoretical and experimental results pertaining to electrokinetics of particle covered surfaces are presented. Theoretical models are discussed, enabling a quantitative evaluation of the streaming current and the streaming potential as a function of particle coverage and their surface properties (zeta potential). Experimental data related to electrokinetic characteristics of particle monolayers, mostly streaming potential measurements, are presented and interpreted in terms of the above theoretical approaches. These results, obtained for model systems of monodisperse colloid particles are used as reference data for discussion of experiments performed for polyelectrolyte and protein covered surfaces. The utility of the electrokinetic measurements for a precise, in situ determination of particle and protein monolayers at various interfaces is pointed out.

Identification of Maize dwarf mosaic virus in Maize in Poland.

From 2005 to 2007 in Southern Wielkopolska, Lower Silesia, and Malopolska regions, maize (Zea mays) plants showing leaf mosaic and stunting symptoms were found. ELISA tests using commercial polyclonal antisera against Maize dwarf mosaic virus (MDMV) obtained from Bioreba (Basel, Switzerland) and Loewe (Munich, Germany) gave positive results in 71 samples. However, the ELISA response for symptomatic plants, in most cases, was low, with OD values ranging from 0.05 to 0.18. Therefore, only eight plants with relatively high virus concentration were chosen for further identification assays. Examination of leaf extracts with an electron microscope revealed the presence of potyvirus-like particles. Symptomatic leaves were positive for MDMV by using immunosorbent electron microscopy (ISEM) with antiserum raised against the Spanish isolate of MDMV (supplied as positive MDMV control from A. Achon, Centre Vdl-Irta, Lleida, Spain). A set of test plants, including sweet corn, dent corn, sorghum (Sorghum vulgare), and true millet (Panicum miliaceum), were mechanically inoculated with extracts from symptomatic plants in 0.05 M phosphate buffer plus 1% ß-mercaptoethanol. Inoculated plants developed symptoms typical of MDMV in 2 to 5 weeks (1,2). For further investigations, three virus isolates were chosen. To confirm the identification of MDMV, reverse transcription (RT)-PCR was performed with total RNA isolated from infected plants with primers 3MDF (5' GAT GAG TTR AAY GTY TAT GCA CGA C 3'), a forward primer in the 3' region of NIb gene and either 1MDR (5' RTG CAT RAT TTG TCT GAA AGT TGG 3') or 3MDR (5' ACC AVA CCA TYA TWC CAC TC 3'), reverse primers in the 3' region of the coat protein gene (A. Zare, Shiraz University, personal communication). 3MDF corresponds to nucleotides 8306 to 8330, 3MDR is complementary to nucleotides 8791 to 8813, and 1MDR is complementary to nucleotides 8917 to 8939 of the MDMV genome (GenBank Accession No. AJ001691). The RT-PCR products obtained were analyzed by agarose gel electrophoresis. Amplicons of the expected sizes (635 and 560 bp) were obtained with RNA from symptomatic plants, but not from asymptomatic plants. The sequence of the 576-bp PCR product was deposited in GenBank (Accession No. EU240460). In alignments done with BlastN ( www.ncbi.nlm.nih.gov/blast ), the highest nucleotide sequence identities were 99% with Spanish MDMV isolates ("SP" AM110758, "SP" AJ416645, and "S1" AJ416635), 91% with the Hungarian isolate "Sc/H, sweet corn" AJ542536, 90% with "MDMV-A" U07216, and 87% with an Israeli MDMV (AF395135). On the basis of these findings, the virus isolated from diseased maize plants was identified as MDMV. The significance of MDMV detection is noteworthy because maize has become an important crop in Poland in recent years and acreage is increasing systematically. References: (1) M. A. Achon et al. Eur. J. Plant Pathol. 102:697, 1996. (2) A. J. Gibbs. Maize dwarf mosaic virus. Page 752 in: Viruses of Plants. Descriptions and Lists from the VIDE database. A. A. Brunt et al., eds. CAB International, Wallingford, UK, 1996.

Rotation due to hydrodynamic interactions between two spheres in contact.

We analyze the rotational and translational motion of two close spheres in a fluid at low Reynolds number to investigate if their surfaces come into mechanical contact. The rotational motion of a sphere settling close to another fixed ball is calculated from a model in which contact interactions between the spheres are added to the gravitational and hydrodynamic forces. The model predicts a transition from pure rolling to rolling with slip, determined by the Coulomb's law, when the ratio of the mechanical friction to the load increases up to the static friction coefficient. The dependence of the angular and translational velocities on the kinetic friction coefficient and on the separation between the particle surfaces is analyzed. The angular and translational velocities of a millimeter size bead in a viscous oil, close to a fixed bead of a similar size, are measured from video images. Interferometric data on translational motion are also collected according to the method introduced in our earlier studies. A systematic fitting procedure of the model to the experiment is developed and applied to the rotational and translational measurements. The model parameters are determined.

Kinetic mechanisms of rat polymerase beta-ssDNA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (Pol beta)(16) and (Pol beta)(5) ssDNA binding mode.

Kinetics of rat polymerase beta (pol beta) binding to the single-stranded DNA (ssDNA) in the (pol beta)(16) and (pol beta)(5) binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol beta)(16) binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol beta, and with a 10-mer which binds the enzyme exclusively in the (pol beta)(5) binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol beta)(16) binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: [formula-see text] A similar mechanism is observed in the formation of the (pol beta)(5) binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol beta)(16) binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol beta, prior to ssDNA binding.

Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase beta in its different ssDNA binding modes.

The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.

Recognition of template-primer and gapped DNA substrates by the human DNA polymerase beta.

Interactions between human DNA polymerase beta and the template-primer, as well as gapped DNA substrates, have been studied using quantitative fluorescence titration and analytical ultracentrifugation techniques. In solution, human pol beta binds template-primer DNA substrates with a stoichiometry much higher than predicted on the basis of the crystallographic structure of the polymerase-DNA complex. The obtained stoichiometries can be understood in the context of the polymerase affinity for the dsDNA and the two ssDNA binding modes, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotide residues occluded by the protein in the complex. The analysis of polymerase binding to different template-primer substrates has been performed using the statistical thermodynamic model which accounts for the existence of different ssDNA binding modes and has allowed us to extract intrinsic spectroscopic and binding parameters. The data reveal that the small 8 kDa domain of the enzyme can engage the dsDNA in interactions, downstream from the primer, in both (pol beta)(16) and (pol beta)(5) binding modes. The affinity, as well as the stoichiometry of human pol beta binding to the gapped DNAs is not affected by the decreasing size of the ssDNA gap, indicating that the enzyme recognizes the ssDNA gaps of different sizes with very similar efficiency. On the basis of the obtained results we propose a plausible model for the gapped DNA recognition by human pol beta. The enzyme binds the ss/dsDNA junction of the gap, using its 31 kDa domain, with slight preference over the dsDNA. Binding only to the junction, but not to the dsDNA, induces an allosteric conformational transition of the enzyme and the entire enzyme-DNA complex which results in binding of the 8 kDa domain with the dsDNA. This, in turn, leads to the significant amplification of the enzyme affinity for the gap over the surrounding dsDNA, independent of the gap size. The presence of the 5'-terminal phosphate, downstream from the primer, has little effect on the affinity, but profoundly affects the ssDNA conformation in the complex. The significance of these results for the mechanistic model of the functioning of human pol beta is discussed.

Energetics and specificity of Rat DNA polymerase beta interactions with template-primer and gapped DNA substrates.

Interactions between rat polymerase beta (pol beta) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol beta complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA downstream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol beta for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol beta. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5' terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.

Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA.

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.

Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: quantitative analysis using the rapid quench-flow method.

The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined by:¿eqalign¿¿¿rm Helicase+ATP¿&¿mathop¿¿rightleftharpoons¿ ¿k_1¿_¿k_¿-1¿¿¿¿rm (H-ATP)¿¿mathop¿¿rightleftharpoons¿ ¿k_2¿_¿k_¿-2¿¿¿¿rm (H-ADP¿cdot Pi)¿¿cr &¿mathop¿¿rightleftharpoons¿ ¿k_3¿_¿k_¿-3¿¿¿¿rm (H-ADP¿cdot Pi)¿ *¿The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP)=(1.3+/-0.5)x10(5) M(-1). The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H)=k(2)/k(-2) approximately 2 while the subsequent conformational transition is characterized by K(3)=k(3)/k(-3) approximately 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release.The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter sigma=0.25(+/-0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed.

Interactions of Escherichia coli replicative helicase PriA protein with single-stranded DNA.

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.

Escherichia coli replicative helicase PriA protein-single-stranded DNA complex. Stoichiometries, free energy of binding, and cooperativities.

Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.

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

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

Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics.

Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, depsilonA(pepsilonA)(19), that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer depsilonA(pepsilonA)(9), which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. This mechanism is described by the equation: Helicase+ssDNAk1/(k1)<-->(k-1)(H-ssDNA)1(k2)<-->(k-2)(H-ssDNA)2 (k3)<-->(k-3)(H-ssDNA)3. The value of the bimolecular rate constant, k(1), is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)(1). These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed.

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.

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.