Internal Motions of Transfer RNA: A Study of Exchanging Protons by Magnetic Resonance36

Abstract

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr.

After dilution of tRNA-H₂O samples in D₂O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region.

Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CGI 1 of yeast tRNA^Phe at 17°C, or 5 minutes for U8-A14 of yeast tRNA^Asp at 20°C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five.

The exchange rates of other,fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA^Phe has a lifetime of 14 ms at 17°C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem.

Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer.

From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether shortlived or long-lived.

Buffer effects have also been observed in poly(rA)-poly(rU), for which we derive a lifetime of 2.5 ms at 27°C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.     

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.     

Proton exchange and base-pair kinetics of poly(rA).poly(rU) and poly(rI).poly(rC)

Proton exchange of poly(rA).poly(rU) and poly(rI).poly(rC) has been studied by nuclear magnetic resonance line broadening and saturation transfer from H2O. Five exchangeable peaks are observed. They are assigned to the imino, amino and 2'-OH ribose protons. The aromatic spectrum is also assigned. Contrary to previous observations, we find that the exchange of the imino proton is strongly buffer sensitive. This property is used to derive the base-pair lifetime, which is in the range of milliseconds at 27 degrees C, 100 times smaller than published values. The enthalpy for the base-opening reaction (-86 kJ/mol) and the insensitivity of the reaction to magnesium suggest that the open state involves a small number of base-pairs. The similarities in the exchange from the two duplexes indicate that the same open state is responsible for exchange of purine and pyrimidine imino protons. For the lifetime of the open state and for the base-pair dissociation constant, we obtain only lower limits. At 27 degrees C they are three microseconds and 10(-3), respectively. The analysis that yields the much larger values published previously is based on the assumption that amino protons exchange only from open base-pairs. But theory and preliminary experiments indicate that it may occur from the closed duplex. The exchange of amino protons is slower than that of the imino protons. Exchange of the 2'-OH protons from the duplexes is much slower than from single-stranded poly(rU), and it is accelerated by magnesium. This could indicate hydrogen-bonding to backbone phosphate. Discrepancies between our results and those of previous studies are discussed.     

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.     

Nuclear magnetic resonance study of the proton exchange rate in the operator-promoter DNA sequence of the trp operon of Escherichia coli

The dynamic behavior of a palindromic oligonucleotide (C-G-T-A-C-T-A-G-T-T-A-A-C-T-A-G-T-A-C-G) representative of the operator sequence and containing the Pribnow box of the trp operon of Escherichia coli was investigated. The resonances of the imino protons and of the H2 protons of the adenosine residues were all assigned. The opening rate constants of the base-pairs were calculated by monitoring the exchange rate of the observable imino protons (nine out of ten), using selective temperature (T1) measurements, which avoid the complication of cross-relaxation and spin diffusion. These measurements have to be performed in conditions where the exchange process is much faster than the opening and closing of the base-pairs, so that the observed exchange rate is equal to the opening rate. It is shown that the catalysis of the exchange process by phosphate dianions is not very efficient (kB approximately equal to 7 X 10(4) M-1 S-1). Hence, in phosphate buffer, the necessary opening-rate limiting condition is met only at high pH values (approximately equal to 9.5) where efficient catalysis by OH- takes place, or at very high buffer concentration. While G X C base-pairs show very little exchange, acting in the sequence as molecular "staples", the A X T base-pairs that are protected from the fraying have very different opening and closing rates, depending on the sequence. Although it seems possible that the opening process could occur at the base-pair level, it is localized at most to two base-pairs in that particular sequence. The activation energies for the opening process of all non-fraying base-pairs are very similar (19 +/- 1 kcal mol-1; 1 cal = 4.184 J), and the differences in the opening rates are essentially due to differences in the activation entropies. With regard to the role of this sequence in the promoter, it is observed that the end of the Pribnow box exchanges relatively easily, and that the activation parameters for the "breathing" process and for the isomerization step of the promoter--RNA polymerase are not very different.     

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.     

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.     

Processes of base-pair opening and proton exchange in Z-DNA

Using proton magnetic resonance, we have investigated imino and amino proton exchange in the Z form of the four oligomers d(Cbr8GCGCbr8G), d(CGm5CGCG), d(CG)6, and d(CG)12. In the latter two oligomers, all five exchangeable protons have been assigned. We find that proton acceptors such as NH3 or the basic form of Tris enhance imino proton exchange. The base-pair lifetime can then be obtained by extrapolation of the exchange time to infinite concentration of proton acceptor. For d(CG)6 and d(CG)12, the values are ca. 3.5 ms at 80 degrees C and ca. 130 ms at 35 degrees C. The latter value is about 65 times longer than in the same oligomers in the B form. The activation energy of base-pair opening, 80 kJ/mol, is the same in the Z and the B forms of d(CG)12. At 5 degrees C, the base-pair lifetime is about 3 s, much smaller than the time constant of the Z to B transition, to which it is therefore unrelated. The base-pair dissociation constant at 35 degrees C, 0.5 X 10(-6), is 5 times smaller than for the same oligomers in the B form. In the absence of added catalyst, at pH 7, the exchange time of the imino proton is 30 min at 5 degrees C. That of both cytidine amino protons, assigned by NOE, is about 50 min. The longest proton exchange time, ca. 330 min, is assigned unambiguously to the guanosine amino protons. Thus assigned and interpreted in terms of exchange chemistry rather than structural kinetics, the exchange times do not support earlier models of Z-DNA internal motions.     

Proton exchange and internal motions in two chromomycin dimer-DNA oligomer complexes.

Previous structural studies on the complexes of the chromomycin (CHR) dimer with duplexes of d(A1-A2-G3-G4-C5-C6-T7-T8) and of d(A1-G2-G3-A4-T5-C6-C7-T8) in solution [one Mg(II) and two drugs per duplex] are extended to hydrogen exchange measurements. Exchange of the OH8 proton of chromomycin, measured by real time proton-deuterium exchange, is very slow and requires dissociation of the complex, whose lifetime is thus determined. The lifetimes and apparent dissociation constants of base pairs are deduced from the catalysis of imino proton exchange by ammonia. The four central base pairs, which interact with the CHR chromophores in the minor groove (Gao & Patel, 1990), may open within the complex, but the opening rate is less than in the free duplex by one to two orders of magnitude. The activation energy for base-pair opening and the differences between the lifetimes of adjacent pairs suggest that single base-pair opening is the predominant imino proton exchange pathway in all cases. In the symmetrical complex of chromomycin with the first duplex, the lifetimes of the central base pairs (G3.C6 and G4.C5) are in the same range (52 and 29 ms, respectively, at 38 degrees C). In the asymmetrical complex formed with the second duplex, the base-pair lifetimes in the G2-G3-A4-T5 segment that interacts with the chromophore moiety are strongly increased. That of G3.C6 is particularly long. Above 50 degrees C, exchange of the G3 imino proton is opening limited.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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).     

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)     

A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions.

The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.     

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.     

1H nuclear magnetic resonance study of the dynamic properties of the B and Z-forms of poly[d(A-br5C).d(G-T)]

Poly[d(A-br5C).d(G-T)], a synthetic polynucleotide with a 50% A-T base composition, undergoes a reversible, highly co-operative transition between the right-handed B and left-handed Z conformations. The latter is stabilized at both elevated temperature and ionic strength. The B and Z-forms of poly[d(A-br5C).d(G-T)] coexist in 4.6 M-NaCl at 45 degrees C. Due to slow exchange, two sets of Tim and Gim resonances are observed and can be assigned to the B and Z conformations (the chemical shifts are, respectively, Tim = 13.4, 14.1 p.p.m. (parts/million); and Gim = 11.9, 12.4 p.p.m.). Measurements of the 1H spin-lattice (R1) and spin-spin (R2) relaxation rates of the exchangeable thymine (Tim) and guanine (Gim) imino protons have been used to probe the internal dynamics of the B and Z-forms of poly[d(A-br5C).d(G-T)] and the mechanism of the B-Z transition. The proton exchange behavior in the B and Z conformations is quite different. At elevated temperature, R1 for both Tim and Gim in the B conformation is dominated by exchange with the solvent, with Tim exchanging more rapidly than Gim. This demonstrates that exchange involves the opening of single base-pairs and that neighboring A-T and G-br5C base-pairs exchange independently of each other. B-form poly[d(A-br5C).d(G-T)] is unusual in that there is an acceleration of the Tim exchange rate with increasing NaCl concentration. Conversion to the Z-form by addition of 4.5 M-NaCl dramatically reduces both the Tim and Gim exchange rates (estimated to be less than 2 s-1 at 70 degrees C). Thus, the G-br5C base-pair and, in particular, the A-T base-pair are stabilized in the Z conformation. By measuring relaxation rates at 45 to 50 degrees C where the B and Z-forms are in equilibrium, we find that the B-Z interconversion rates are less than two per second. In the B conformation at 25 degrees C, the dipolar contributions to the imino proton relaxation rates are about one-third of those expected on the basis of a rigid rod model for 65 base-pair fragments, a difference we assign to large amplitude (30 degrees high frequency (less than 100 ns) out-of-plane motions of the bases. Conversion to the Z conformation has little effect on the dipolar contributions to relaxation, i.e. on the internal motions.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.