A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids

A helix-destabilizing protein, HD40 (Mr 40,000), isolated from the cytoplasm of Artemia salina (Marvil, D.K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472) stoichiometrically disrupts the secondary structures of synthetic single-stranded and helical polynucleotides (e.g. poly(rA), poly(dA), poly(rC), poly(dC), and poly(rU)) as well as those of natural polynucleotides (e.g. MS2 RNA and phi X174 viral DNA). The conformations of double-stranded DNA and double- or triple-stranded synthetic polynucleotides are not affected by the protein. Formation of duplexes, e.g. poly(rA . rU), is prevented by HD40 at 25 to 50 mM but not at 100 to 140 mM NaCl. The unwinding of the residual secondary structure of RNA and DNA by HD40 is not highly cooperative and has a stoichiometry of one HD40 per 12 to 15 nucleotides. The addition of HD40 in excess of 1 molecule per 12 to 15 nucleotides results in the cooperative formation of distinct bead-like structures along the nucleic acid strand. The beads are about 20 nm in diameter with a center to center distance of about 40 nm. The appearance of the beads is not accompanied by any spectral changes (CD and UV) beyond those obtained at a stoichiometry of one HD40 molecule per 12 to 15 nucleotides.     

Characterization of a mitochondrial protein binding to single-stranded DNA.

A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA. The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found. This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA. By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes.     

Binding of RecA protein to single-stranded nucleic acids: spectroscopic studies using fluorescent polynucleotides

Binding of the recA gene product from Escherichia coli to single-stranded polynucleotides has been investigated using poly(dA) that have been modified by chloroacetaldehyde to yield fluorescent 1,N6-ethenoadenine (epsilon A) bases. A strong enhancement of the fluorescent quantum yield of poly(d epsilon A) is induced upon RecA protein binding. A 4-fold increase is observed in the absence of ATP or ATP gamma S and a 7-fold increase in the presence of either nucleoside triphosphate. RecA protein can bind to poly(d epsilon A) in the absence of both Mg2+ ions and ATP (or ATP gamma S) but Mg2+ ions are required to observe RecA protein binding in the presence of ATP (or ATP gamma S) at pH 7.5. ATP binding to the RecA-poly(d epsilon A) complex induces a dissociation of RecA from the polynucleotide followed by re-binding of [RecA-ATP-Mg2+] ternary complex. Whereas ATP-induced dissociation of RecA-poly(d epsilon A) complexes is a fast process, the subsequent binding reaction of [RecA-ATP-Mg2+] is slow. A model is proposed whereby [RecA-ATP-Mg2+] binding to poly(d epsilon A) involves slow nucleation and elongation processes along the polynucleotide backbone. The nucleation reaction is shown to involve at least a trimer or a tetramer. Polymerization of the [RecA-ATP-Mg2+] ternary complex stops when the polynucleotide is entirely covered with 6 +/- 1 nucleotides per RecA monomer. ATP hydrolysis then induces a release of RecA-ADP complexes from the polynucleotide template.     

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.     

A pulse-radiolysis study of the reaction of hydrated electrons with N'-formylkynurenine and related compounds: electron-transfer reactions with nucleic acid components.

The reactivity of N'-formylkynurenine (FK) derivatives towards eaq has been investigated. The reduced transient species have been characterized (lambda max approximately 340, 440 nm, epsilon lambda max approximately 3000-1000 M-1 cm-1, pKa approximately 7.8). Owing to the strong FK electron affinity, electron-transfer reactions occur from purine (except guanine) and pyrimidine electron adducts to FK (k approximately 2-7 x 10(9) M-1 s-1). As some FK derivatives bind to DNA (or polynucleotides) the protective effect of complexation on FK-DNA (or polynucleotides) adduct formation has been investigated.     

Adsorption of single-stranded and double-helical polynucleotides on the mercury electrode

The adsorption of single-stranded polynucleotides and double-helical DNA on the dropping mercury electrode has been studied with the aid of Breyer's alternating current (a.c.) polarography. Our results indicate that all three constituents of polynucleotides (residues of bases, sugar, and phosphoric acid) are involved in the adsorption. At neutral pH their participation in adsorption depends on the ionic strength, the potential of the electrode, and the conformation of the polynucleotide in the solution. At an ionic strength of about 0.1, double-helical DNA is adsorbed electrostatically on a positively charged electrode surface by inadequately masked negative charges of the phosphate groups. At a higher ionic srength (about 0.5), this electrostatic adsorption is no longer detectable by using a.c. polarography; under these conditions it is probable that native DNA is adsorbed around the potential of the electrocapillary maximum with the aid of sugar residues and a few bases. Single-stranded polynucleotides, on the other hand, are primarily adsorbed by means of the bases. Desorption of double-helical DNA occurs around a potential of ?1.2 V against SCE. At this potential, the helical regions of single-stranded polynucleotides are also desorbed. Desorption of the disordered regions of single-stranded polynucleotides occurs at more negative potentials. Adsorption and desorption of a small number of bases released from double-helical DNA was evident in the a.c. polarograms only at elevated temperature, or at room temperature after degradation of DNA by sonication.     

Structure-spectrum correlations in nucleic acids. I. Raman lines in the 600-700 cm-1 range of guanosine residue.

Raman spectra of nine crystals of known structures which involve guanosine moieties with various conformations have been observed. It has been established that a guanosine residue with the C3'endo-anti conformation gives a strong Raman line at 666 +/- 2 cm-1. It has also been found that the residue with 04'endo-anti gives a strong Raman line at 682 cm-1, and C3'exo-syn at 616 cm-1. The usefulness of these structure-spectrum correlations in the conformation studies of polynucleotides are shown.     

Multiple species of single-stranded nucleic acid-binding proteins in Saccharomyces cerevisiae

DNA affinity chromatography has been used to identify the major single-stranded nucleic acid binding proteins (SSBs) of Saccharomyces cerevisiae. There are five abundant species having molecular masses of 50, 45, 31, 23, and 20 kDa. Four of these proteins are cytoplasmic and one is mitochondrial. To date, three of the proteins have been purified to homogeneity. The purified proteins are designated SSB-m, SSB-1, and SSB-2, with molecular masses of 20, 45, and 50 kDa, respectively. SSB-m is found only in mitochondrial subcellular fractions. SSB-1 stimulates purified yeast DNA polymerase I, while SSB-2 inhibits DNA polymerase I. An antibody against SSB-1 has been prepared in rabbits and purified by SSB-1-Sepharose affinity chromatography. The purified antibody specifically inhibits DNA synthesis in an in vitro replication system, suggesting that SSB-1 may be involved in DNA replication in vivo. SSB-2 has the highest affinity for single-stranded DNA of all three proteins. It may represent a new class of eukaryotic SSB, on the basis of molecular weight, inhibition of DNA polymerase and antigenicity. Antibodies have also been prepared against SSB-2. The immunological reagents have been used to show that SSB-1, SSB-2, and SSB-m are antigenically distinct, as well as to study the relationship of these three SSBs to other proteins in yeast.     

Spatial configuration of single-stranded polynucleotides. Calculations of average dimensions and Nmr coupling constants

The probability distributions of the torsional angles (Φ′, ω′, ω, Φ, and ψ), which fix the structure of nucleotide backbone, have been calculated using the results of energy calculations based on extended Huckel theory (EHT), complete neglect of differential overlap (CNDO), perturbative configuration interaction using localized orbitals (PCILO), and classical potential functions (CPF) methods. Statistical average values of the vicinal ¹H? ¹H, ¹H? ³¹P, and ¹³C? ³¹P nmr coupling constants 〈J〉 have been calculated from the generalized Karplus relations using the probability distribution in the Φ′, Φ, and ψ space. Experimental 〈J〉 values for polyribouridylic acid (polyU) support the theoretical predictions for these torsional angles. Using Monte Carlo technique, random coils of single-stranded polynucleotides have been simulated and the mean-square end-to-end distance 〈r²〉 has been calculated. Molecular orbital methods (EHT, CNDO, and PCILO) suggest considerable flexibility around O? P bonds, leading to fairly small values for the characteristic ratio (C_∞ ～ 4). Observed values of the unperturbed characteristic ratio for polynucleotides are quite large (C_∞ ～ 18) suggesting a relatively rigid nucleotide backbone. The results based on molecular orbital calculations can be reconciled with the experimental values by introducing an additional stabilization of ～2 kcal mol^?1 for the predicted minimum energy ragion (Φ′ ～ 240°, ω′ ～ 290°, ω 290°, Φ 180°, and ψ 60°). Such a stabilization may arise from the association of water molecules and metal ions with the phosphate group and (or) Coulomb interaction between neighboring phosphate groups. The calculations provide a semiquantitative estimate of torsional rigidity in the nucleotide backbone.     

Interactions of aromatic residues of proteins with nucleic acids. Fluorescence studies of the binding of oligopeptides containing tryptophan and tyrosine residues to polynucleotides.

The binding of oligopeptides of general structure Lys-X-Lys (where X is an aromatic residue) to several polynucleotides has been studied by fluorescence spectroscopy. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups as shown by the ionic strength and pH dependence of binding. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the nucleic acid bases with the aromatic amino acid whose fluorescence is quenched. Fluorescence data have been quantitatively analyzed according to a model involving these two types of complexes. Association constants and the size of binding sites have been determined. Stacking interactions are favored in single-stranded polynucleotides as compared to double-stranded ones. A short oligopeptide such as Lys-X-Lys is thus able to distinguish between single-stranded and double-stranded nucleic acids. Fluorescence results are compared to those obtained by proton magnetic resonance and circular dichroism.     

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.     

Stereochemistry of nucleic acids and polynucleotides. VI. Minimum energy conformations of dimethyl phosphate

As part of a study on the conformation of polynucleotides and nucleic acids the preferred conformations of the model conpound dimethyl phosphate are worked out using potential energy functions. In calculating the total potential energy associated with the conformation, nonbonded, torsional, and electrostatic terms have been considered. The variation of the total conformational energy is represented as a function of two torsion angles ? and ψ which are the rotations about the two phosphoester bonds. The most stable conformations are found to be the gauche–gauche conformations about these bonds. The conformations observed for phosphodiesters in the solid state and in the proposed structures of polynucleotides and nucleic acids cluster around the minimum. Also, regions of minimum energy correspond well with the typical allowed regions of a representative dinucleotide.     

Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.     

Spin labeled nucleic acids.

Homopolyribonucleotides and E. coli DNA wer spin labeled with an iodoacetamide-nitroxide compound. The extent of labeling is highly dependent upon the nature of the base and the secondary structure of the nucleic acid. This spin label-polymer linkage is unstable at high temperatures and in phosphate buffers. In order to determine the effect of changes in the environment of nucleic acids on the esr signals of their attached spin labels, the polynucleotides were subjected to temperature and viscosity perturbations. An increase in temperature (T) affects a linear decrease in the anisotropy factor of the esr signal. The log tau (tau = correlation time) versus (1/T) profile is linear with a positive slope when the spin label is attached to single stranded polynucleotides but exhibits discontinuities at certain critical temperatures when attached to the duplexes poly (As-U) and poly (I-Cs). These critical temperatures are lower than the optical Tm. Logarithmic increase in viscosity was found to produce a linear increase in tau in aqueous sucrose solutions.     

Block-units method for conformational calculations of large nucleic acid chains. II. The two-hierarchical approach and its application to conformational arrangement of the unusual T psi C loop of rabbit tRNA(Val)

The two-level hierarchical methodology is suggested for conformational calculations of large fragments of nucleic acids. The method of the first level is intended for performing a fast screening of the conformational phase space. The high-level method may be used to refine structurally important conformations. The method of the first level is the block-units method, which has been developed specially for these purposes (see part I). It has been shown that the block-units method allows the satisfactory calculation of the structure parameters of the optimal conformations of polynucleotides. The results of the conformational rearrangement calculations of the T psi C loop of the tRNA(Phe) after modification of its sequence are represented.     

Interaction of nucleic acids with electrically charged surfaces: II. Conformational changes in double-helical polynucleotides

The influence of adsorption of double-stranded (ds) DNA, ds RNA and homopolymeric pairs at a mercury electrode on conformation of these polynucleotides was studied. Changes in the polarographic reducibility of polynucleotides, which were followed by means of normal pulse polarography and linear sweep peak voltammetry at the dropping mercury electrode were exploited to indicate conformational changes. It was found that, as a consequence of adsorption of ds polynucleotides on the negatively charged electrode conformational changes similar to denaturation take place in a narrow potential region around ?1.2 V (the region U). After sufficiently long time of the contact with the electrode (under our conditions about 10 s) these changes reach limiting values, which can approach total denaturation. Upon adsorption of ds polynucleotides on the electrode charged to more positive potentials than the region U either (1) no conformational changes occur or (2) only a small part of the polynucleotide (probably labile regions of the ds molecule) is very quickly denatured - the remainder of the molecule preserves its ds structure. Conformational changes of adsorbed ds polynucleotides are influenced by factors which change the stability of ds polynucleotides in solution. It is supposed that denaturation of ds polynucleotides in the region U might result from the strains connected with the repulsion of certain segments of the molecule anchored on the electrode from the negatively charged surface.     

Polyelectrolyte theory of charged-ligand binding to nucleic acids

The Poisson-Boltzmann framework provides simple and sound formulae for evaluation of the interactions of polynucleotides with charged ligands. The theory is based on the observation that shape-dependent effects are small. Hence the results for the charged plane may be used as a first appromixation for polyelectrolytes. The counterion distribution and the extent of counterion or ligand binding is derived on the basis of a sumrule for counterion concentrations, combined with the mass-action law. Calculations require no more than a pocket calculator, even in the case of mixed-salt solutions. Significant qualitative results are given, and applied to the problems of site-binding and of repressor-DNA interaction.     

Matrix-method calculation of linear and circular dichroism spectra of nucleic acids and polynucleotides

Calculations of the optical properties (absorption, linear dichroism, circular dichroism, and anisotropic components of the CD) are presented for polynucleotides of random or regular sequence within the formalism of the matrix method using a set of parameters that includes only the ππ* transitions of the aromatic bases. Experimental solution spectra agree favorably with calculated CD spectra for A-RNA, A-DNA, and B-DNA, when coordinates derived from x-ray studies on fibers are used. Excessive hypochromicity is predicted when parameters intended to reproduce the vacuum-uv absorption of the chromophores are included in the calculations, but total elimination of these parameters leads to an insufficient hypochromicity for the long-wavelength absorption band. Using alternative conformations for DNA in low-salt aqueous solution did not improve the agreement between experimental and calculated spectra, but some features of the optical properties predicted for these variant structures suggest that the tilt of the bases with respect to the helical axis may be larger than that of the fiber B-form. In the case of polynucleotides with regular structure, which have been traditionally less easy to understand in terms of the standard nucleic acid conformations, a series of alternative structures has been examined. Unexpectedly, the calculated spectrum for the Z-DNA structure compares almost quantitatively with the experimental spectrum of poly(dGC·dGC) in low salt. This result, which confirms a recent report [Vasmel, H. & Greve, J. (1981) Biopolymers 20 , 1329–1332], is in contrast with the current identification of Z-DNA with the high-salt form of poly(dGC·dGC). Finally, the optical properties of single-stranded polyribonucleotides appear to be better explained when alternative structures [9₁-helix for poly(rA) and 6₁-helix for poly(rC)] are introduced instead of the A-RNA form.     

Conformational geometry and vibrational frequencies of nucleic acid chains.

Normal coordinate analysis of diethyl phosphate has been made, which predicts all observed Raman frequencies in the range 170–1300 cm^?1. The force constants from this calculation have been transferred to a vibrational calculation for a simplified model of the backbone of nucleic acids, which also involves the ? O? PO₂^?? O phosphate group and the ? C₅′? C₄′? C₃′? linkage of the ribose. The coordinates of these atoms are those recently given by Arnott and Hukins, which place the ribose ring of B-DNA in a C₃′-exo conformation. This simple polymer model appears to be able to describe adequately the frequency-dependent changes observed in the Raman spectra arising from the backbone vibrations of nucleic acid in going from the B- to A-form. The symmetric ? O? P? O? diester stretch increases in frequency from about 787 cm^?1 in the B-form to 807 cm^?1 in the A-form. The increased frequency characteristic of the A-form is due to the combining of the diester stretch with vibrations involving the C₅′, C₄′, and C₃′ nuclei. The frequency of the symmetric ? O? P? O? diester stretch is shown to be very dependent on the conformation of the ribose ring, indicating that in polynucleotides the ribose ring takes on one of two rigid conformations: C₃′-endo for A-form or C₃′-exo for B-form and “disordered” polynucleotides. The calculation lends confirmation to the atomic coordinates of Arnott and Hukins since the use of other geometries with the same force constants failed to give results in agreement with experimental evidence. The calculations also demonstrate the lowering effect of hydration on the anionic PO stretching frequencies. Experimental results show that the 814-cm^?1 band observed in the spectra of 5′GMP gel arises from a different vibrational mode than that of the 814-cm^?1 band of A-DNA.     

p53-catalyzed annealing of complementary single-stranded nucleic acids.

p53 has been reported to inhibit the DNA helicase intrinsic to simian virus 40 large tumor antigen (T antigen). We found that inhibition is not restricted to T antigen, but also affects several other DNA and RNA helicases. Complexing of the helicases by the p53 protein as a possible inactivation mechanism could be excluded. Instead, the anti-helicase activity can be explained by our finding that p53 binds with high affinity to single-stranded nucleic acids and has a strong DNA.DNA and RNA.RNA annealing activity. We could also show that p53 is able to alter the secondary structure of RNA and/or to influence dynamic RNA-RNA interactions. These results, and the fact that the affinity of p53 to RNA is about one order of magnitude higher than to single-stranded DNA, imply an RNA-specific function of p53 in vivo.