Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.     

Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR.

Integration Host Factor, IHF, is an E. coli DNA binding protein that imposes a substantial bend on DNA. Previous footprinting studies and bending assays have characterized several recognition sequences in the bacterial and lambda phage genome as unique in the way they are bound by IHF. We have chosen one of the lambda phage sites, H1, for study because it presents a small yet sequence-specific substrate for NMR analysis of the complex. A 19 base-pair duplex, H19, corresponding to the recognition sequence at the H1 site was constructed by isotopically labeling one of the strands with 15N. (1H, 15N) heteronuclear NMR experiments aided in assigning the imino proton resonances of the DNA alone and in complex with IHF. The NMR results are consistent with a mode of binding observed in the recent crystal structure of IHF bound to another of its sites from the lambda phage genome. Additionally, the dramatic change that IHF imposes on the imino proton chemical shifts is indicative of a severe deviation from canonical B-DNA structure. In order to understand the dynamic properties of the DNA in the complex with IHF, the exchange rates of the imino protons with the solvent have been measured for H19 with and without IHF bound. A drastic reduction in exchange is observed for the imino protons in the IHF bound DNA. In the DNA-protein complex, groups of adjacent base-pair exchange at the same rate, and appear to close more slowly than the rate of imino proton exchange with bulk water, since their exchange rate is independent of catalyst concentration. We infer that segments of the double helix as large as 6 bp open in a cooperative process, and remain open much longer than is typical for opening fluctuations in naked duplex DNA. We discuss these results in terms of the specific protein-DNA contacts observed in the crystal structure.     

Imino proton exchange and base-pair kinetics in RNA duplexes

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton, we have measured the base-pair lifetimes and the dissociation constants of six RNA duplexes: [r(CGCGAUCGCG)](2), [r(CGCGAAUUCGCG)](2), [r(CCUUUCGAAAGG)](2), [r(CGCACGUGCG)](2), [r(GGU(8)CC).r(GGA(8)CC)], and [poly(rA).poly(rU)], and we compare them with those of their DNA homologues. As predicted by a two-state (closed/open) model of the pair, the imino proton exchange times decrease linearly vs. the inverse of catalyst concentration. As in DNA duplexes, base pairs open one at a time, and the kinetics is in most cases insensitive to the nature of the adjacent residues. The lifetime of the r(G.C) pairs, 40 to 50 ms, is longer than that of the equivalent in the corresponding oligodeoxynucleotides, and the dissociation constants, about 10(-)(7), are slightly smaller. The r(A.U) opening and closing rates are much larger than those of the d(A.T) pairs, but the stabilities are comparable.     

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.     

Proton exchange in DNA-luzopeptin and DNA-echinomycin bisintercalation complexes: rates and processes of base-pair opening.

Imino proton exchange studies are reported on the complexes formed by bisintercalation of luzopeptin around the two central A.T pairs of the d(CCCATGGG) and d(AGCATGCT) duplexes and of echinomycin around the two central C.G pairs of the d(AAACGTTT) and d(CCAAACGTTTGG) duplexes. The depsipeptide backbone of the drugs occupies the minor groove of the complexes at the bisintercalation site. The exchange time of the amide protons of the depsipeptide rings provides a lower estimate of the complex lifetime: 20 min at 15 degrees C for the echinomycin complexes and 4 days at 45 degrees C for the luzopeptin complexes. The exchange time of imino protons is always shorter than the complex lifetime. Hence, base pairs open even within the complexed oligomers. For the two base pairs sandwiched between the aromatic rings of the drug, the base-pair lifetime is strongly increased, and the dissociation constant is correspondingly reduced. Hence, the lifetime of the open state is unchanged. This suggests similar open states in the free duplex and in the complex. In contrast to the sandwiched base pairs, the base pairs flanking the intercalation site are not stabilized in the complex. Thus, the action of the bisintercalating drug may be compared to a vise clamping the inner base pairs. Analysis suggests that base-pair opening may require prior unwinding or bending of the DNA duplex.     

"Asymmetric" opening reaction mechanism of Z-DNA base pairs: a hydrogen exchange study

With the tritium-Sephadex method, the hydrogen-exchange kinetics of the five NH protons of guanine and cytosine residues in Z-form poly(dG-dC) X poly (dG-dC) were measured as a function of temperature and catalyst concentration. Over the measured temperature range from 0 to 34 degrees C, two classes of protons with constant amplitudes are found. The three protons of the fast class, which were assigned to the guanine amino and imino protons, have an exchange half-time in the minute time range (at 20 degrees C the half-time is 2.5 min) and an activation energy of 18 kcal mol-1. Since these two types of protons exchange at the same rate in spite of their grossly different pK values, the exchange of these protons must be limited by the same nucleic acid conformational change. The two cytosine amino protons of the slow class are especially slow with exchange half-times in the hour time range (at 20 degrees C the exchange half-time is 1 h) and the activation energy is 20 kcal mol-1. The exchange of these two protons is not limited by some nucleic acid conformational change as shown by the marked exchange acceleration of these protons upon addition of 0.2 M imidazole. In addition, we have also reexamined the hydrogen-deuterium exchange kinetics of the amino protons of guanosine cyclic 2',3'-monophosphate by a spectral difference method using a stopped-flow spectrophotometer. The measured kinetic process is monophasic with a rate constant of 3 s-1 at 20 degrees C, which is in the same range as the predicted rate constant of the guanine amino protons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton exchange and base-pair opening kinetics in 5''-d(CGCGAATTCGCG)-3'' and related dodecamers.

We have used nuclear magnetic resonance (NMR) spectroscopy to measure the lifetimes of individual base-pairs in the palindromic DNA oligonucleotide 5'-d(CGCGAATTCGCG)-3' and in three other dodecamers with symmetrical base substitutions in the sites underlined. The resonances of the hydrogen-bonded imino protons in each of the substituted oligomers in the duplex form have been assigned using one dimensional nuclear Overhauser effect (1-D NOE) experiments. The lifetimes have been obtained from the dependence of selective longitudinal relaxation times and linewidths of the imino proton resonances on the concentration of base catalyst (Tris) at 25 degrees C and in the presence of 50 mM NaCl. The lifetimes of the central A.T base-pairs have been found to depend on base sequence. They are greatly increased in the dodecamer 5'-d(CGCAAATTTGCG)-3' which contains an A3T3 tract. The lifetimes of the central A.T base-pairs in 5'-d(CGCGAATTCGCG)-3', 5'-d(CGCTAATTAGCG)-3' and 5'-d(CGCCAATTGGCG)-3' are comparable. In all dodecamers, the lifetime of the A.T base-pair at the 5'-end of the AnTn tract is the shortest. The anomalous opening kinetics of the A.T base-pairs can be correlated to the bending properties of the corresponding sequences.     

Structural energetics and base-pair opening dynamics in sarcin-ricin domain RNA

The sarcin-ricin domain is a universal element of the RNA from the large ribosomal subunit. The domain is part of the binding site for elongation factors and is specifically cleaved by the toxins alpha-sarcin and ricin. In this work, we have mapped the energetics and dynamics of individual structural motifs in a 29-mer RNA oligomer containing the sarcin-ricin domain. The stability of individual base pairs in the structure was characterized from measurements of the exchange rates of imino protons using nuclear magnetic resonance spectroscopy at 10 degrees C. The measurements also provided the rates of opening and closing for selected base pairs. The results reveal that the structural stabilization free energies in the sarcin-ricin domain are broadly distributed between 2.9 and 10.6 kcal/mol. One of the least stable sites in the structure is the noncanonical G-A base pair located next to the phosphodiester bond that is cleaved by alpha-sarcin. The low stability of this base pair supports the proposal that cleavage by alpha-sarcin occurs by a base flipping mechanism. The opening dynamics of other base pairs is affected by elements of the structure such as the bulged-G motif and its cross-strand stacking. Participation in these motifs increases the lifetimes of the bases in an open, solvent-accessible conformation.     

Enhanced base-pair opening in the adenine tract of a RNA double helix

Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the kinetics and energetics of base-pair opening in two nucleic acid double helices. One is the RNA duplex 5'-r(GCGAUAAAAAGGCC)-3'/5'-r(GGCCUUUUUAUCGC)-3', which contains a central tract of five AU base pairs. The other is the homologous DNA duplex with a central tract of five AT base pairs. The rates and the equilibrium constants of the opening reaction of each base pair are measured from the dependence of the exchange rates of imino protons on ammonia concentration, at 10 °C. The results reveal that the tract of AU base pairs in the RNA duplex differs from the homologous tract of AT base pairs in DNA in several ways. The rates of opening of AU base pairs in RNA are high and increase progressively along the tract, reaching their largest values at the 3'-end of the tract. In contrast, the opening rates of AT base pairs in DNA are much lower than those of AU base pairs. Within the tract, the largest opening rate is observed for the AT base pair at the 5'-end of the tract. These differences in opening kinetics are paralleled by differences in the stabilities of individual base pairs. All AU base pairs in the RNA are less stable than the AT base pairs in the DNA. The presence of the tract enhances these differences by increasing the stability of AT base pairs in DNA while decreasing the stability of AU base pairs in RNA. Due to these divergent trends, along the tracts, the AU base pairs become progressively less stable than AT base pairs. These findings demonstrate that tracts of AU base pairs in RNA have specific dynamic and energetic signatures that distinguish them from similar tracts of AT base pairs in DNA.     

Effects of sequence and length on imino proton exchange and base pair opening kinetics in DNA oligonucleotide duplexes.

The base catalysed imino proton exchange in DNA oligonucleotides of different sequences and lengths was studied by 1H-NMR saturation recovery experiments. The self-complementary sequences studied were GCGCGAATTCGCGC (I), CGCGAATTCGCG (II), GCGAATTCGC (III), and CGCGATCGCG (IV). The evaluation of base pair lifetimes was made after correction for the measured 'absence of added catalyst' effect which was found to be characterized by recovery times of 400-500 ms for the AT base pairs and 250-300 ms for the GC base pairs at 15 degrees C. End effects with rapid exchange is noticeable up to 3 base pairs from either end of the duplexes. The inner hexamer cores GAATTC of sequences I-II show similar base pair lifetime patterns, around 30 ms for the innermost AT, 5-10 ms for the outer AT and 20-50 ms for the GC base pairs at 15 degrees C. The shorter sequences III and particularly IV show much shorter lifetimes in their central AT base pairs (11 ms and 1 ms, respectively).     

Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A-tracts

We have investigated by NMR the effects of NH(4)(+) on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B-DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](2), the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH(4)(+) binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH(4)(+) concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH(4)(+) concentration is kept larger than 1 M, the A.T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH(3) proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (W?rml?nder, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).     

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, HPO4(2-) or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2.G23 base pair was the most stable base pair in all the duplexes. The conserved U-1*G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2'-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1*G22, C-2*G23 and A2*U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed.     

Analysis of proton exchange kinetics with time-dependent exchange rate

Mass spectrometry is used to probe the kinetics of hydrogen–deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange. The data are described by a power-like function which is based on a time-dependence of the exchange rate. Experimental data ranging over many time scales is considered and accurate fits of a power-like function are obtained. Results of fittings show correlation between faster hydrogen–deuterium exchange and increase of pH. Furthermore a model is presented that discriminates between easily exchangeable hydrogens (located in close proximity to the protein surface) and those protected from the exchange (located in the protein interior). A possible interpretation of the model and its biological significance are discussed.     

Measurement of the helix opening rate in Z-DNA by 1H nuclear magnetic resonance relaxation spectroscopy

The exchange rate of the hydrogen-bonded guanine imino protons N(1) in the high-salt form of Poly(dG-dC) was measured by following the non-selective inversion-recovery of their 1H NMR signal at 360 MHz, in the temperature range between 77 degrees C and 90 degrees C. In a 4.5M NaCl solution, Poly(dG-dC) is believed to adapt the left-handed Z-conformation, and the results reported here represent the first quantitative measurements of this rate process for Z-DNA by Nuclear Magnetic Resonance, complementing previous measurements made by tritium exchange at 0 degrees C (Ramstein, J. and Leng, M. (1980) Nature 288, 413-414). The results confirm that this process is much slower in the Z-form, compared to the B-structure, and that this difference in rates results mainly from a large decrease in the entropy of activation for Z-DNA.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Cross-relaxation bottleneck in water-lysozyme proton magnetization exchange

The proton spin-lattice relaxation parameters in natural and deuterated lysozyme solutions have been measured as a function of temperature (0-50 degrees C). The variation of the apparent magnitudes of the water proton magnetizations in the solutions with temperature indicates that magnetic coupling mixes protein and water proton magnetizations. The results are consistent with an exchange cross-relaxation model (Hills, B. P., Mol Phys 1992, 76, 489-508) in which the cross-relaxation acts between the labile and nonlabile protons, rather than between water and protein protons. Although this cross-relaxation pathway clearly affects the observed magnetization fractions in this protein solution, its influence on the relaxation rates is less apparent.     

Direct estimation of base-pair exchange kinetics in oligo-DNA by a combination of NOESY and ROESY experiments.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (< or = 20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and pi ex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fast- and slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.     

Proton exchange and base-pair lifetimes in a deoxy-duplex containing a purine-pyrimidine step and in the duplex of inverse sequence

Using proton relaxation and magnetization transfer from water we have measured the imino proton exchange kinetics in two dodecadeoxynucleotide duplexes. One is formed by the self-complementary sequence 5'-d(C-C-T-T-T-C-G-A-A-A-G-G), the other by the inverse sequence. The imino proton exchange rates are found to depend on the concentration of ammonia or imidazole, acting as basic catalysts of proton exchange. Extrapolation of exchange times to infinite catalyst concentration yields the base-pair lifetimes, for instance 40 milliseconds for the central G.C base-pair of the 5'-d(C-C-T-T-T-C-G-A-A-A-G-G) duplex and four milliseconds for its A.T neighbour, at 15 degrees C. These results differ markedly from those reported by other laboratories for similar deoxy compounds. An explanation of the discrepancy has been proposed recently. Differences between base-pair lifetimes indicate that opening is not co-operative. From the catalyst efficiency relative to exchange from isolated nucleosides, we estimate the dissociation constant of each base-pair, e.g. 0.3 x 10(-6) and 1.5 x 10(-5) at 15 degrees C, for the same G.C and A.T base-pairs. The lifetime and dissociation constant of corresponding base-pairs of the two duplexes are similar, except for the central G.C base-pair. This correlates with differences in the solution structures reported by others. We have completed the assignments of the imino protons and of the six cytosine amino protons of the 5'-d(G-G-A-A-A-G-C-T-T-T-C-C) 12-mer. A new base-pair numbering scheme is proposed.     

Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal sister chromatid exchange

An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPC-11 between 3' regions of the C gamma 2a and C gamma 2b heavy chain genes results in duplication of the C gamma 2a heavy chain gene and generation of a novel recombination joint. The USCE occurs between (TC)n tracts adjacent to alternating purine-pyrimidine tracts. We have investigated the capacity of both the donor regions and the recombinant product involved in this event to adopt left-handed Z-DNA and intramolecular triplexes. The results of chemical probing with diethylpyrocarbonate and osmium tetroxide at the base pair level demonstrate that under the influence of negative supercoiling the alternating purine-pyrimidine regions of these plasmids can adopt Z-DNA at neutral pH, and the oligopurine.oligopyrimidine (pur.pyr) regions of these regions can adopt intramolecular triplexes at low pH (less than or equal to pH 6.0). At intermediate pH values, mixtures of both structures are present. Increasing the negative superhelical density of the plasmid does not increase the amount of triplex present at neutral pH indicating that the presence of long Z-DNA segments adjacent to pur.pyr tract prevents intramolecular triplex formation. In summary, we conclude that the sequences involved in the USCE can form either an intramolecular triplex in the (TC)n tract or Z-DNA in the alternating purine-pyrimidine tract and that Z-DNA will predominate under physiological conditions. The presence of segments which adopt Z-DNA at a site of USCE suggests that formation of this structure may enhance recombination between adjacent pur.pyr tracts.