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.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Cation binding linked to a sequence-specific CAP-DNA interaction

The equilibrium association constant observed for many DNA-protein interactions in vitro (K(obs)) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that release of cations from the DNA is the dominant involvement of ions in the binding reaction. With this assumption, the graph of logK(obs) versus log[MX] is predicted to have a constant slope proportional to the number of ions released from the DNA upon protein binding. However, experimental data often deviate from log-linearity at low salt concentrations. Here we show that for the sequence-specific interaction of CAP with its primary site in the lactose promoter, ionic stoichiometries depend strongly on cation identity and weakly on anion identity. This outcome is consistent with a simple linkage model in which cation binding by the protein accompanies its association with DNA. The order of ion affinities deduced from analysis of DNA binding is the same as that inferred from urea-denaturation experiments performed in the absence of DNA, suggesting that ion binding to free CAP contributes significantly to the ionic stoichiometry of DNA binding. In living cells, the coupling of ion-uptake and DNA binding mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.     

ELECTRON STAINS : I. Chemical Studies on the Interaction of DNA with Uranyl Salts

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO₂⁺⁺/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ⅓. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.     

Equilibrium dissociation and unfolding of the Arc repressor dimer

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.     

The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA

The equilibrium dissociation constant of the DNA binding domain of interferon regulatory factor 1 (IRF1 DBD) for its DNA binding site depends strongly on salt concentration and salt type. These dependencies are consistent with IRF1 DBD binding to DNA, resulting in the release of cations from the DNA and both release of anions from the protein and uptake of a cation by the protein. We demonstrated this by utilizing the fact that the release of fluoride from protein upon complex formation does not contribute to the salt concentration dependence of binding and by studying mutants in which charged residues in IRF1 DBD that form salt bridges with DNA phosphates are changed to alanine. The salt concentration dependencies of the dissociation constants of wild-type IRF1 DBD and the mutants R64A, D73A, K75A, and D73A/K75A were measured in buffer containing NaF, NaCl, or NaBr. The salt concentration and type dependencies of the mutants relative to wild-type IRF1 DBD provide evidence of charge neutralization by solution ions for R64 and by a salt bridge between D73 and K75 in buffer containing chloride or bromide salts. These data also allowed us to determine the number, type, and localization of condensed ions around both IRF1 DBD and its DNA binding site.     

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.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.     

Chromotin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication.

A Xenopus homologue of Schizosaccharomyces pombe cdc21 has been characterized as a new member of the MCM family of proteins. The cdc21 protein exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 binds to decondensing chromatin at the end of mitosis, localizing to numerous foci which form prior to reconstitution of the nuclear membrane. The association of cdc21 with chromatin occurs in membrane-free high speed extracts and is resistant to detergent extraction. The spatial organization of the cdc21 foci resembles that of pre-replication centres though no co-localization with RP-A was observed. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced as the DNA replication forks progress. These subnuclear changes in localization correlate with cell-cycle-regulated changes in phosphorylation. Cdc21 binds to chromatin in an underphosphorylated state, but in early S phase the nuclear localized cdc21 is partially phosphorylated before it is displaced from the chromatin. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor.     

Thermodynamic analysis of the single-stranded DNA binding activity of the archaeal replication protein A (RPA) from Sulfolobus solfataricus

The single-stranded DNA binding protein from Sulfolobus solfataricus (Sso-RPA) binds single-stranded DNA with dissociation constants in the range of 10-30 nM at room temperature. The affinity for DNA decreases at higher temperatures. At 85 degrees C, the optimal growth temperature of the crenarchaeot S. solfataricus, the dissociation constant is only about 1 microM. We analyzed the equilibrium between Sso-RPA and a fluorescently labeled 13 nucleotide oligonucleotide by fluorescence anisotropy measurements in the presence of four different salts and in the temperature range between 10 and 60 degrees C. In the presence of potassium chloride and choline chloride, three to four ions are released upon complexation, independent of the temperature. In contrast, in the presence of potassium fluoride and potassium glutamate, we observed a significant change of the number of ions released when the temperature was varied. The binding reaction is strongly exothermic with enthalpies of about -55 to -70 kJ/mol, depending upon the salt. Van't Hoff analysis suggests that the binding enthalpy is temperature independent.     

Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.     

Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.     

Temperature and salt concentration alter base-sequence selectivity of a duplex DNA-binding protein

A structural polymorphism of nucleic acids, which depends on the concentration of cations and the conditions of hydration, are strongly involved with interactions between DNA and proteins. In this paper, we report that different DNA sequences bound to hyperthermostable TATA-box-binding protein (PhoTBP) at different combinations of temperature and salt concentration in in vitro selection experiments. As a result of the interaction of-these selected DNAs with PhoTBP, characteristic changes in the numbers of water molecules and ions occurred under each condition of the selection experiment. This finding could help us to understand the solvent environment-dependent preference for base sequences in protein–DNA interactions.