Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G)

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.     

1H NMR determination of base-pair lifetimes in oligonucleotides containing single base mismatches

Proton nuclear magnetic resonance (NMR) spectroscopy is employed to characterize the kinetics of base-pair opening in a series of 9mer duplexes containing different single base mismatches. The imino protons from the different mismatched, as well as fully matched, duplexes are assigned from the imino-imino region in the WATERGATE NOESY spectra. The exchange kinetics of the imino protons are measured from selective longitudinal relaxation times. In the limit of infinite exchange catalyst concentration, the exchange times of the mismatch imino protons extrapolate to much shorter lifetimes than are commonly observed for an isolated GC base pair. Different mismatches exhibit different orders of base-pair lifetimes, e.g. a TT mismatch has a shorter base-pair lifetime than a GG mismatch. The effect of the mismatch was observed up to a distance of two neighboring base pairs. This indicates that disruption in the duplex caused by the mismatch is quite localized. The overall order of base-pair lifetimes in the selected sequence context of the base pair is GC > GG > AA > CC > AT > TT. Interestingly, the fully matched AT base pair has a shorter base-pair lifetime relative to many of the mismatches. Thus, in any given base pair, the exchange lifetime can exhibit a strong dependence on sequence context. These findings may be relevant to the way mismatch recognition is accomplished by proteins and small molecules.     

Altered structural fluctuations in duplex RNA versus DNA: a conformational switch involving base pair opening

DNA and RNA are known to have different structural properties. In the present study, molecular dynamics (MD) simulations on a series of RNA and DNA duplexes indicate differential structural flexibility for the two classes of oligonucleotides. In duplex RNA, multiple base pairs experienced local opening events into the major groove on the nanosecond time scale, while such events were not observed in the DNA simulations. Three factors are indicated to be responsible for the base opening events in RNA: solvent-base interactions, 2'OH(n)-O4'(n+1) intra-strand hydrogen bonding, and enhanced rigid body motion of RNA at the nucleoside level. Water molecules in the major groove of RNA contribute to initiation of base pair opening. Stabilization of the base pair open state is due to a 'conformational switch' comprised of 2'OH(n)-O4'(n+1) hydrogen bonding and a rigid body motion of the nucleoside moiety in RNA. This rigid body motion is associated with decreased flexibility of the glycosyl linkage and sugar moieties in A-form structures. The observed opening rates in RNA are consistent with the imino proton exchange experiments for AU base pairs, although not for GC base pairs, while structural and flexibility changes associated with the proposed conformational switch are consistent with survey data of RNA and DNA crystal structures. The possible relevance of base pair opening events in RNA to its many biological functions is discussed.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Influence of the polyamines spermine and spermidine on yeast tRNAPhe as revealed from its imino proton NMR spectrum.

A comparison of imino proton NMR spectra of yeast tRNAPhe recorded at various solution conditions indicates, that polyamines have a limited effect on the structure of this tRNA molecule. Polyamines are found to catalyse the solvent exchange of several imino protons in yeast tRNAPhe not only of non hydrogen bonded imino protons, but also of imino protons of the GU and of some AU and tertiary base pairs. It is concluded that at low levels of catalysing components the exchange rates of the latter protons are not determined by the base pair lifetime. In the presence of high levels of spermidine the solvent exchange rates of imino protons of several base pairs in the molecule were assessed as a function of the temperature. Apparent activation energies derived from these rates were found to be less than 80 kJ/mol, which is indicative for (transient) independent opening of the corresponding base pairs. In the acceptor helix the GU base pair acts as a dynamic dislocation. The AU base pairs at one side of the GU base pair exhibit faster transient opening than the GC base pairs on the other side of this wobble pair. The base pairs m2GC10 and GC11 from the D stem and GC28 from the anticodon stem show relatively slow opening up to high temperatures. Model studies suggest that 1-methyladenosine, an element of tRNA itself, catalyses imino proton solvent exchange in a way similar to polyamines.     

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).     

Imino proton exchange in DNA catalyzed by ammonia and trimethylamine: evidence for a secondary long-lived open state of the base pair

The base-pair opening kinetics of the self-complementary oligomer d(CGCGAATTCGCG)(2) has been derived from NMR measurements of the imino proton exchange. In general, it has previously been found that imino proton exchange in duplex DNA is limited by the proton-transfer step from the open state and that the dependence of the exchange times on the inverse concentration of an added exchange catalyst is linear. In the present study, a curvature is observed for, in particular, the innermost AT base pair with both ammonia and trimethylamine (TMA) as exchange catalysts. The two catalysts act on the same open states, but the accessibility of TMA is reduced by a factor of 2-3 compared to ammonia. Assuming that ammonia accesses the imino proton equally in the open state of the base pair and in the mononucleoside, the curvature is consistent with 7-9% of the openings ending in open states with lifetimes of about 1 micros while the bulk of open-state lifetimes fall in the nanosecond range. A curvature is also found for the exchange times of the imino protons in the A-tract sequence CGCA(8)CGC/GCGT(8)GCG. This curvature becomes increasingly pronounced from the 5'-end toward the center of the tract and hereby seems to be correlated with the contraction of the minor groove. Thus, while the base-pair lifetimes deduced from the present study are in accordance with previous measurements, a substantial fraction of the open states formed by the central AT-base pairs in the two oligomers exhibits microsecond lifetimes in contrast to previous estimates in the nanosecond range. These findings may be of relevance for the way sequence specific recognition is accomplished by proteins and ligands.     

Effects of the presence of an aldehydic abasic site on the thermal stability and rates of helix opening and closing of duplex DNA.

The presence of an abasic site in duplex DNA lowers the thermodynamic stability, as monitored by the optical melting temperature, and decreases the rate of imino proton exchange with water, by about an order of magnitude, as monitored by direct measurement of both the exchange lifetimes and the imino proton T1S. The exchange lifetimes of the imino protons with water as a function of base catalyst concentration were analyzed to determine the origin of the effect of the abasic site on imino exchange lifetimes. Analysis of the results showed that the helix opening rate is not significantly changed by the presence of an abasic site. The differences in exchange lifetimes are attributed to a faster helix closing rate in the presence of an abasic site. The faster rate of helix closing may be an important contribution to the stability of abasic sites in duplex DNA to base-catalyzed elimination reaction. It is noted that duplex DNAs containing analogues of the aldehydic abasic site apparently do not exhibit these exchange lifetime effects.     

Dynamic opening of DNA during the enzymatic search for a damaged base

Uracil DNA glycosylase (UDG) removes uracil from U.A or U.G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.     

Base pair opening in three DNA-unwinding elements

DNA-unwinding elements are specific base sequences that are located in the origin of DNA replication where they provide the start point for strand separation and unwinding of the DNA double helix. In the present work we have obtained the first characterization of the opening of individual base pairs in DNA-unwinding elements. The three DNA molecules investigated reproduce the 13-mer DNA-unwinding elements present in the Escherichia coli chromosome. The base sequences of the three 13-mers are conserved in the origins of replication of enteric bacterial chromosomes. The exchange of imino protons with solvent protons was measured for each DNA as a function of the concentration of exchange catalyst using nuclear magnetic resonance spectroscopy. The exchange rates provided the rates and the equilibrium constants for opening of individual base pairs in each DNA at 20 degrees C. The results reveal that the kinetics and energetics of the opening reactions for AT/TA base pairs are different in the three DNA-unwinding elements due to long range effects of the base sequence. These differences encompass the AT/TA base pairs that are conserved in various bacterial genomes. Furthermore, a qualitative correlation is observed between the kinetics and energetics of opening of AT/TA base pairs and the location of the corresponding DNA-unwinding element in the origin of DNA replication.     

Kinetics and energetics of base-pair opening in 5'-d(CGCGAATTCGCG)-3' and a substituted dodecamer containing G.T mismatches.

Proton nuclear magnetic resonance (NMR) spectroscopy is used to characterize the kinetics and energetics of base-pair opening in the dodecamers 5'-d(CGCGAATTCGCG)-3' and 5'-d(CGCGAATTTGCG)-3'. The latter dodecamer contains two symmetrical G.T mismatched base pairs. The exchange kinetics of imino protons is measured from resonance line widths and selective longitudinal relaxation times. For the G.T pair, the two imino protons (G-N1H and T-N3H) provide probes for the opening of each base in the mismatched pair. The lifetimes of individual base pairs in the closed state and the equilibrium constants for formation of the open state are obtained from the dependence of the exchange rates on the concentration of ammonia catalyst. The activation energies and standard enthalpy changes for base-pair opening are obtained from the temperature dependence of the lifetimes and equilibrium constants, respectively. The results indicate that the G.T mismatched pairs are kinetically and energetically destabilized relative to normal, Watson-Crick base pairs. The lifetimes of the G.T pairs are of the order of 1 ms or less, over the temperature range from 0 to 20 degrees C. The equilibrium constants for base-pair opening, at 20 degrees C, are increased up to 4000-fold, relative to those of normal base pairs. The energetic destabilization of the G.T base pairs is, at least in part, enthalpic in origin. The presence of the G.T mismatched base pairs destabilizes also neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.     

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.     

Premelting thermal fluctuational interbase hydrogen-bond disrupted states of a B-DNA guanine-cytosine base pair: significance for amino and imino proton exchange.

Modified self-consistent phonon theory when applied to the DNA double helix indicates the existence of fairly long-lived states in which single interbase H bonds are disrupted. One can then postulate a number of situations in which particular disrupted H bonds can enhance particular proton exchange. In this paper we postulate a number of such partially open states for a B-conformation GC base pair and calculate the probability of each of these states for a B-conformation poly(dG).poly(dC). We compare these probabilities to those probabilities needed to explain various observed proton exchange rates. We propose that, for a GC base pair in B conformation, there are two amino proton exchangeable states--a cytosine amino proton exchangeable state and a guanine amino proton exchangeable state; both require the disruption of only the corresponding interbase H bond. The imino proton exchange, however, requires the disruption of all three interbase H bonds and this defines a third open state. Our calculated probabilities for a GC base pair in these three states are in fair agreement with available experimental estimates from measurements of amino and imino proton exchange.     

An NMR study of A-T base pair opening rates in oligonucleotides. Influence of sequence and of adenine methylation.

We report relaxation time measurements by semi-selective and totally selective NMR techniques on the thymidine imino protons of d(GGATATCC) and d(GGm6ATATCC). For these oligonucleotides helix fraying, rather than single base pair opening, is the major exchange mechanism even 25 degrees C below the Tm. We have therefore applied a new saturation transfer technique to measure exchange rates at temperatures where fraying has a very small or negligible contribution. Measurements of exchange rates as a function of temperature give significantly different activation energies for base pairs 3 and 4 in d(GGATATCC). Adenine methylation results in a slowing down of the opening rate for the m6A-T base pair but surprisingly has an even greater effect upon the adjacent non-methylated A-T base pair.     

Evidence from base-pair kinetics for two types of adenine tract structures in solution: their relation to DNA curvature

We have measured the base-pair lifetimes in oligodeoxynucleotides containing tracts of A.T base pairs using imino proton magnetic resonance. When the tract contains more than four consecutive A.T base pairs, possibly including a 5'-AT step but not a 5'-TA step, anomalously long lifetimes are observed. For example, the lifetimes of the central A.T base pairs of the dodecamer 5'-d-CGCAAAAAAGCG are 122 and 91 ms at 15 degrees C whereas, in the same conditions, the lifetime of the central A.T pair of the decamer 5'-d-CGCGATCGCG is only 4 ms, a value similar to those measured in several other B-DNA oligoduplexes [Leroy et al. (1988) J. Mol. Biol. 200, 223-238]. This strongly suggests that, in tracts of four A.T pairs or more, a conformation distinct from standard B-DNA is formed cooperatively. All sequences known to generate curved DNA exhibit anomalously long base-pair lifetimes. This is the first local and physical property shown to correlate with DNA curvature. Our observations suggest that the structure responsible for the long lifetimes is involved in the curvature of DNA.     

Base-pair dynamics in an antiparallel DNA triplex measured by catalyzed imino proton exchange monitored via 1H NMR spectroscopy

Using (1)H NMR spectroscopy, the base-pair opening dynamics of an antiparallel foldback DNA triplex and the corresponding duplex has been characterized via catalyzed imino proton exchange. The triplex system was found to be in an equilibrium between a duplex and a triplex form. The exchange rate between the two forms (i.e., the on/off-rate of the third strand) was measured to be 5 s(-1) at 1 degrees C, and the base-pair dynamics of both forms were investigated separately. Both Watson-Crick and reverse Hoogsteen base pairs were found to have base-pair lifetimes in the order of milliseconds. The stability of the Watson-Crick base pairs was, however, substantially increased in the presence of the third strand. In the DNA triplex, the opening dynamics of the reverse Hoogsteen base pairs was significantly faster than the dynamics of the Watson-Crick pairs. We were able to conclude that, for both Watson-Crick and reverse Hoogsteen base pairs, spontaneous and individual opening from within the closed base triplet is the dominating opening pathway.     

Probing the role of hydrogen bonds in the stability of base pairs in double-helical DNA

Aromatic stacking and hydrogen bonding between nucleobases are two of the key interactions responsible for stabilization of DNA double-helical structures. The present work aims at defining the specific contributions of these interactions to the stability of individual base pairs in DNA. The two DNA double helices investigated are formed, respectively, by the palindromic base sequences 5'-dCCAACGTTGG-3' and 5'-dCGCAGATCTGCG-3'. The strength of the N==H...N inter-base hydrogen bond in each base pair is characterized from the measurement of the protium-deuterium fractionation factor of the corresponding imino proton using NMR spectroscopy. The structural stability of each base pair is evaluated from the exchange rate of the imino proton, measured by NMR. The results reveal that the fractionation factors of the imino protons in the two DNA double helices investigated fall within a narrow range of values, between 0.92 and 1.0. In contrast, the free energies of structural stabilization for individual base pairs span 3.5 kcal/mol, from 5.2 to 8.7 kcal/mol (at 15 degrees C). These findings indicate that, in the two DNA double helices investigated, the strength of N==H...N inter-base hydrogen bonds does not change significantly depending on the nature or the sequence context of the base pair. Hence, the variations in structural stability detected by proton exchange do not involve changes in the strength of inter-base hydrogen bonds. Instead, the results suggest that the energetic identity of a base pair is determined by the number of inter-base hydrogen bonds, and by the stacking interactions with neighboring base pairs.     

Base pair opening dynamics of a 2-aminopurine substituted Eco RI restriction sequence and its unsubstituted counterpart in oligonucleotides.

Studies of 1H NMR selective saturation recovery were performed to determine the imino proton exchange with solvent water of the base pairs in the Eco RI endonuclease recognition sequence GAATTC, placed at the center of self-complementary decamer and dodecamer oligonucleotides. In one oligonucleotide the innermost adenine was replaced by the fluorescent base analogue 2-aminopurine (2AP). From the measurements at different concentrations of TRIS buffer acting as proton exchange catalyst, base pair lifetimes were evaluated. The results at 25 degrees show that the AT base pairs have lifetimes of the order of a few ms, whereas the surrounding GC base pairs in a dodecamer have lifetimes of about 100 ms. The (2AP)T base pair has a shorter lifetime than the corresponding AT base pair. The temperature dependent optical absorption, and for the 2AP containing oligonucleotide fluorescence, were used to study the single strand-duplex equilibrium of the decamers. The results indicate that NMR and the optical techniques, although applied at very different concentrations, monitor the same conformational transition of the oligonucleotide.