Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Structural equilibria in RNA as revealed by 19F NMR

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.     

UV spectroscopic identification and thermodynamic analysis of protonated third strand deoxycytidine residues at neutrality in the triplex d(C(+)-T)6:[d(A-G)6.d(C-T)6]; evidence for a proton switch.

Near-UV difference spectral analysis of the triplex formed from d(C-T)6 and d(A-G)6.d(C-T)6 in neutral and acidic solution shows that the third strand dC residues are protonated at pH 7.0, far above their intrinsic pKa. Additional support for ion-dipole interactions between the third strand dC residues and the G.C target base pairs comes from reduced positive dependence of triplet stability on ionic strength below 0.9 M Na+, inverse dependence above 0.9 M Na+ and strong positive dependence on hydrogen ion concentration. Molecular modeling (AMBER) of C:G.C and C+:G.C base triplets with the third strand base bound in the Hoogsteen geometry shows that only the C+:G.C triplet is energetically feasible. van't Hoff analysis of the melting of the triplex and target duplex shows that between pH 5.0 and 8.5 in 0.15 M NaCl/0.005 M MgCl2 the enthalpy of melting (delta H degree obs) varies from 5.7 to 6.6 kcal.mol-1 for the duplex in a duplex mixture and from 7.3 to 9.7 kcal.mol-1 for third strand dissociation in the triplex mixture. We have extended the condensation-screening theory of Manning to pH-dependent third strand binding. In this development we explicitly include the H+ contribution to the electrostatic free energy and obtain [formula: see text]. The number of protons released in the dissociation of the third strand from the target duplex at pH 7.0, delta n2, is thereby calculated to be 5.5, in good agreement with approximately six third strand dc residues per mole of triplex. This work shows that when third strand binding requires protonated residues that would otherwise be neutral, triplex formation and dissociation are mediated by proton uptake and release, i.e., a proton switch. As a by-product of this study, we have found that at low pH the Watson-Crick duplex d(A-G)6.d(C-T)6 undergoes a transition to a parallel Hoogsteen duplex d(A-G)6.d(C(+)-T)6.     

Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides

Effects of different end sequences on melting, circular dichroism spectra (CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both sides by sequences differing in AT content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from a van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming that the melting transition is two-state. Melting free energies (20 degrees C) evaluated from DSC melting experiments on the four duplex DNAs ranged from -52.2 to -77.5 kcal/mol. Free energies based on the van't Hoff analysis were -37.9 to -58.8 kcal/mol. Although the values are different, trends in the melting free energies of the four duplex DNAs as a function of sequence were identical in both DSC and optical analyses. Subject to several assumptions, values for the initiation free energy were estimated for each duplex, defined as DeltaG(int) = DeltaG(cal) - DeltaG(pred), where DeltaG(cal) is the experimental free energy at 20 degrees C determined from the experimentially measured values of the transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal). The predicted free energy of the sequence, DeltaG(pred)(20 degrees C), is obtained using published nearest-neighbor sequence stability values. For three of the four duplexes, values of DeltaG(int) are essentially nil. In contrast, the duplex with 81.8% GC has a considerably higher estimate of DeltaG(int) = 7.1 kcal/mol. The CD spectra for the six duplexes collected over the wavelength range from 200 to 320 nm are also sequence-dependent. Factor analysis of the CD spectra by singular value decomposition revealed that the experimental CD spectra could be reconstructed from linear combinations of two minor and one major subspectra. Changes in the coefficients of the major subspectrum for different sequences reflect incremental sequence-dependent variations of the CD spectra. Equilibrium binding by BamHI restriction endonuclease to the 40 base pair DNAs whose central eight base pairs contain the recognition sequence for BamHI restriction enzyme bounded by A.T base pairs, 5'-A-GGATCC-A-3' was investigated. Binding assays were performed by titering BamHI against a constant concentration of each of the duplex DNA substrates, in the absence of Mg(2+), followed by analysis by gel retardation. Under the conditions employed, the enzyme binds but does not cleave the DNAs. Results of the assays revealed two binding modes with retarded gel mobilities. Binding isotherms for the fraction of bound DNA species versus enzyme concentration for each binding mode were constructed and analyzed with a simple two-step equilibrium binding model. This analysis provided semiquantitative estimates on the equilibrium binding constants for BamHI to the four DNAs. Values obtained for the binding constants varied only 7-fold and ranged from 6 x 10(-)(8) to 42 x 10(-)(8) M, with binding free energies from -8.6 to -9.7 (+/- 0.2) kcal/mol depending on the sequence that flanks the enzyme binding site. Unlike what was found earlier in binding studies of the 22 base pair duplexes that constitute the core modules of the present 40-mers [Riccelli, P. V., Vallone, P. M., Kashin, I., Faldasz, B. D., Lane, M. J., and Benight, A. S. (1999) Biochemistry 38, 11197-11208], no obvious relationship between binding and stability was found for these longer DNAs. Apparently, effects of flanking sequence stability on restriction enzyme binding may only be measurable in very short duplex deoxyoligonucl     

Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base-pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14).

Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5'G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3' linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation delta Gloop (n) and single-strand circles delta Gcircle (N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy delta H degrees and entropy delta S degrees for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, pp. 377-399).(ABSTRACT TRUNCATED AT 400 WORDS)     

Duplex-tetraplex equilibrium between a hairpin and two interacting hairpins of d(A-G)10 at neutral pH.

d(A-G)10 forms two helical structures at neutrality, at low ionic strength a single-hairpin duplex, and at higher ionic strength a double-hairpin tetraplex. An ionic strength-dependent equilibrium between these forms is indicated by native PAGE, which also reveals additional single-stranded species below 0.3 M Na+, probably corresponding to partially denatured states. The equilibrium also depends upon oligomer concentration: at very low concentrations, d(A-G)10 migrates faster than the random coil d(C-T)10, probably because it is a more compact single hairpin; at high concentrations, it co-migrates with the linear duplex d(A-G)10 x d(C-T)10, probably because it is a two-hairpin tetraplex. Molecular weights measured by equilibrium sedimentation in 0.1 M Na+, pH 7, reveal a mixture of monomer and dimer species at 1 degree C, but only a monomer at 40 degrees C; in 0.6 M Na+, pH 7, only a dimer species is observed at 4 degrees C. That the single- and double-stranded species are hairpin helices, is indicated by preferential S1 nuclease cleavage at the center of the oligomer(s), i.e., the loop of the hairpin(s). The UV melting transition below 0.3 M Na+ or K+, exhibits a dTm/dlog[Na+/K+] of 33 or 36 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex to a single-hairpin duplex with extrahelical residues. When [Na+/K+] > or = 0.3 M, dTm/dlog [Na+/K+] is 19 or 17 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex directly to single strands. A two-hairpin structure stabilized by G-tetrads is indicated by differential scanning calorimetry in 0.15 M Na+/5 mM Mg2+, with deltaH of formation per mole of the two-hairpin tetraplex of -116.9 kcal or -29.2 kcal/mol of G-tetrad.     

Parallel-stranded linear homoduplexes of d(A+-G)n > 10 and d(A-G)n > 10 manifesting the contrasting ionic strength sensitivities of poly(A+.A+) and DNA.

In contrast to shorter homologs which only form a single-stranded nucleic acid alpha-helix in acid solution at [Na+]</=0.02 M Na+, d(A-G)20,30 form in addition a parallel-stranded duplex with (A+.A+) and (G.G) base pairs and interstrand dA+...PO2-ionic and dA+NH2... O=P H-bonds. Under conditions where duplex prevails over alpha-helix, the contribution of the base-backbone interactions to stability varies directly with [H+] and inversely with [Na+], just as in poly(A+.A+). These duplexes are characterized by intense circular dichroism and a large cooperative thermally-induced hyperchromic transition that is dependent on oligomer concentration. Dimethylsulfate reactivity of the dG residues indicates G.G and therefore dA+.dA+rather than dA+.G base pairs. At much higher ionic strength (Na+>/=0.2 M) the protonated base-backbone interactions are so weakened that duplex stability becomes increasingly dependent upon H-bonded base pairing and stacking and almost independent of pH. Between pH 6 and 8 this duplex structure is devoid of protonated dA residues and shows positive dependence of T m on ionic strength similar to that of DNA.     

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.     

Melting and chemical modification of a cyclized self-splicing group I intron: similarity of structures in 1 M Na+, in 10 mM Mg2+, and in the presence of substrate

C IVS is the cyclized form of the intron from the RNA precursor of the Tetrahymena thermophila large subunit (LSU) ribosomal RNA. C IVS was mapped by chemical modification in 1 M Na+, 0.05 M Na+ and 10 mM Mg2+ (Na+/Mg2+), and Na+/Mg2+ with CUCU substrate. The results suggest the secondary structure is similar for all three conditions. Optical melting curves were also measured for C IVS in 1 M Na+ and Na+/Mg2+ and indicate the secondary structures have similar stabilities under both conditions. Computer predictions of secondary structure and stability are in good agreement with observations. The results suggest that many of the approximations used for computer prediction of secondary structure by free energy minimization are reasonable.     

Solvent effects on the stability of A7U7p

The thermodynamics of double-helix formation were measured spectrophotometrically for A7U7 in water at 1 M NaCl and for A7U7p in a variety of solvent mixtures and salt. Comparison of the A7U7 results with calorimetric measurements indicates duplex formation involves intermediate states. For A7U7p between 0.06 and 0.55 M Na+, dTm/d(log [Na+]) = 17.4 degrees C, similar to the value of 19.6 degrees C for poly-(A).poly(U) [Krakauer, H., & Sturtevant, J. M. (1968) Biopolymers 6, 491-512]. At 1 M NaCl, the A7U7p duplex is most stable in 100% water. For 10 mol % solutions, the order for A7U7p duplex stability is ethylene glycol greater than glycerol greater than ethanol greater than 2-propanol greater than dimethyl sulfoxide greater than 1-propanol greater than formamide greater than N,N-dimethylformamide greater than urea greater than dioxane. Comparison of changes in stability and thermodynamic parameters with literature results for proteins suggests proteins and A7U7p interact differently with solvent. The results suggest hydrophobic bonding is not a major contributor to the stability of the A7U7p duplex. Comparisons with bulk solvent surface tension suggest the energy of cavity formation is also not a major contributor to duplex stability.     

The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.     

Melting of oligodeoxynucleotides with various structures

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62-bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end-structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na+] less than 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30-40 bp-long conforms to the two state model. From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na+.     

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.     

Influence of 2-aminoadenosine, A', on the conformational behaviour of d(T-A'-T-A'). A one-dimensional proton NMR study at 300 MHz and 500 MHz

Proton NMR studies at 300 MHz and 500 MHz in aqueous solution were carried out on the modified self-complementary tetranucleoside trisphosphate d(T-A'-T-A'), in which d(A') represents 2-aminodeoxyadenosine. The NMR spectra were observed at two sample concentrations over the temperature range 2-70 degrees C. Assignments, based on homonuclear decoupling and nuclear Overhauser enhancement (NOE) experiments, are given. The concentration dependence of the chemical shift vs temperature profiles was used to extract information concerning duplex formation. The proton-proton and proton-phosphorus coupling constants were obtained at four temperatures and yielded accurate conformational data on the sugar ring and on the back-bone angles beta, gamma and delta. From the observed line broadening, shift profiles, NOEs, and from the observation of imino proton resonances, it is concluded that the compound exists as a miniduplex at low temperature. Comparison of these observations with similar observations on the parent compound d(T-A-T-A) indicate that substitution of dA by dA' increases the tendency towards duplex formation. At low temperature the compound adopts a stacked B-DNA type structure: destacking occurs on raising the temperature. The sequence-dependent sugar ring geometry [Mellema, J.-R., Pieters, J. M. L., van der Marel, G. A., van Boom, J. H., Haasnoot, C. A. G. & Altona, C. (1984) Eur. J. Biochem. 143, 285-301] is present in this molecule. The conformational parameters of d(T-A'-T-A') and d(T-A-T-A) are quite similar, thus substitution of dA by dA' has no measurable influence on the geometry of the sugar ring or on the backbone angles beta, gamma and delta.     

Enthalpy of helix-coil transition of DNA: dependence on Na+ concentration and GC-content

The enthalpy of helix-coil transition of DNA (delta H) is determined from the experiments on DNA melting with ligands by means of absolutely general formula, which contains only values directly known from the experiment (M.D. Frank-Kamenetskii, and A.T. Karapetian, Mol. Biol. USSR 6, 621 (1972)) with the combination of the "area" method (P.O. Vardevanian, et al., Biophysica 28, 130 (1983)). The experimentally obtained data show that delta H depends on both concentration of Na+ in solution and GC-content of DNA and is of high accuracy.     

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures

Melting temperatures, T(m), were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between T(m) and ln [Na(+)] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 degrees C when tested against an independent validation set of T(m) measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this T(m) salt correction is insensitive to DNA concentration. The T(m) salt correction function was found to be sequence-dependent and varied with the fraction of G.C base pairs, in agreement with previous studies of genomic and polymeric DNAs. The salt correction function is independent of oligomer length, suggesting that end-fraying and other end effects have little influence on the amount of sodium counterions released during duplex melting. The results are discussed in the context of counterion condensation theory.     

Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I). d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of M1 and M2 revealed that these fragments form B-DNA type duplex structures. A comparison of the chemical-shift data of the nicked duplexes with those of the intact duplex of III + IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The chemical-shift profiles of M1 and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of M1 were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of M1 and M3 it is concluded that the melting behaviour of M1 occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 degrees C.     

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.     

3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys

[3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers.     

Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches

We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216-17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and beta-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k(cat) for the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant.