Thermodynamic Consequences of an Abasic Lesion in Duplex DNA Are Strongly Dependent on Base Sequence † (original) (raw)
Related papers
Biopolymers, 2018
Bistrand lesions embedded within a single helical turn of tridecameric deoxyoligonucleotide duplexes represent a model system for exploring the impact of clustered lesions that occur in vivo and pose a significant challenge to cellular repair machineries. Such investigations are essential for understanding the forces that dictate lesion-induced mutagenesis, carcinogenesis, and cytotoxicity within a context that mimics local helical perturbations caused by an ionizing radiation event. This study characterizes the structural and energy profiles of DNA duplexes harboring synthetic abasic sites (tetrahydrofuran, F) as models of clustered bistrand abasic (AP) lesions. The standard tridecameric dGCGTACCCATGCGÁdCGCATGGGTACGC duplex is employed to investigate the energetic impact of single and bistrand AP sites by strategically replacing one or two bases within the central CCC/GGG triplet. Our combined analysis of temperature-dependent UV and circular dichroism (CD) profiles reveals that the proximity and relative orientation of AP sites within bistranddamaged duplexes imparts a significant thermodynamic impact. Specifically, 3 0-staggered lesions (CCF/GFG) exert a greater destabilizing effect when compared with their 5 0-counterpart (FCC/ GFG). Moreover, a duplex harboring the central bistrand AP lesion (CFC/GFG) is moderately destabilized yet exhibits distinct properties relative to both the 3 0 and 5 0-orientations. Collectively, our energetic data are consistent with structural studies on bistrand AP-duplexes of similar sequence in which a 3 0-staggered lesion exerts the greatest perturbation, a finding that provides significant insight regarding the impact of orientation on lesion repair processing efficiency.
Thermodynamics of dT−dT Base Pair Mismatching in Linear DNA Duplexes and Three-Arm DNA Junctions †
Biochemistry, 1997
We have used a combination of magnetic-suspension densimetry and calorimetry to derive complete thermodynamic profiles, including volume changes, for the formation of linear DNA duplexes and three-arm branched DNA junctions, from their component strands, with and without dT-dT mismatches. The formation of each type of complex at 20°C is accompanied by a favorable free energy, with a favorable enthalpy term partially compensated by an unfavorable entropy. Formation is associated also with net uptake of water molecules. Using the formation of the fully-paired linear duplex or threearm junction as reference states, we can establish a thermodynamic cycle in which the contribution of the single-strand species cancels. From this cycle, we determine that substitution of dA for dT has a differential free energy of ∆∆G°of +2.4 kcal mol-1 for mismatched duplex and +2.0 kcal mol-1 (on the average) for the mismatched junction. These unfavorable differential free energies result from an unfavorable enthalpy, partially compensated by a favorable entropy, and a negative ∆∆V. The free energies in the two cases have signs opposed to those of ∆∆V, a situation that implicates hydration changes in creating the mismatch. When the ∆∆V terms are normalized by the total number of base pairs involved, the immobilization of structural water molecules (and/or substitution of electrostricted for hydrophobic water molecules) is about 7 times greater for junctions than duplexes. This is consistent with more extensive hydrophobic hydration of branched DNA structures than of duplexes.
Chemical Research in Toxicology, 2000
The T‚G mismatch and the exocyclic adduct 3,N 4 -ethenocytosine ( C) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or C base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T‚G and C-containing 15mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5′-TTXTT, 5′-AAXAA, 5′-CCXCC, or 5′-GGXGG sequences, where X is T, C, or two closely related structural derivatives of C: 3,N 4 -ethanocytosine (EC) and 8-(hydroxymethyl)-C (8-HM-C). Each of the four lesions, incorporated opposite G, decreased both the thermal (T m ) and thermodynamic stability (∆G°3 7 ) of the 15-mer control duplexes. On the basis of the T m and ∆G°3 7 values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-C > EC > C > T‚G. The ∆T m values range from -15.8 to -9.5°C when C t ) 8 µM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2°C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4°C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3.0°C
Nucleic Acids Research, 2010
The thermodynamic contributions of rA • dA, rC • dC, rG • dG and rU • dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the "G 37 , "H and melting temperature (T m) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4 C, respectively. The general trend in decreasing stability for the single internal mismatches is rG • dG > rU • dT > rA • dA > rC • dC. The stability trend for the base pairs 5 0 of the single internal mismatch is rG • dC > rC • dG > rA • dT > rU • dA. The stability trend for the base pairs 3 0 of the single internal mismatch is rC • dG > rG • dC >> rA • dT > rU • dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST.
Biochemistry, 2009
Locked nucleic acids (LNA), conformationally restricted nucleotide analogues, are known to enhance pairing stability and selectivity toward complementary strands. With the aim to contribute to a better understanding of the origin of these effects, the structure, thermal stability, hybridization thermodynamics, and base-pair dynamics of a full-LNA:DNA heteroduplex and of its isosequential DNA:DNA homoduplex were monitored and compared. CD measurements highlight differences in the duplex structures: the homoduplex and heteroduplex present B-type and A-type helical conformations, respectively. The pairing of the hybrid duplex is characterized, at all temperatures monitored (between 15 and 37°C), by a larger stability constant but a less favorable enthalpic term. A major contribution to this thermodynamic profile emanates from the presence of a hairpin structure in the LNA single strand which contributes favorably to the entropy of interaction but leads to an enthalpy penalty upon duplex formation. The base-pair opening dynamics of both systems was monitored by NMR spectroscopy via imino protons exchange measurements. The measurements highlight that hybrid G-C base-pairs present a longer base-pair lifetime and higher stability than natural G-C base-pairs, but that an LNA substitution in an AT base-pair does not have a favorable effect on the stability. The thermodynamic and dynamic data confirm a more favorable stacking of the bases in the hybrid duplex. This study emphasizes the complementarities between dynamic and thermodynamical studies for the elucidation of the relevant factors in binding events. Locked nucleic acids (LNA), 1 conformationally restricted nucleotide analogues, constitute an important addition to the tools available for nucleic acid diagnostics and nucleic acid therapeutics. LNA monomers contain a modified ribose moiety in which the 2 0 O and 4 0 C are linked by a methylene bridge (2 0-O,4 0-C-methylene-β-D-ribofuranosyl), locking the sugar in the C3 0-endo/N-type conformation (1-3). LNA resemble natural nucleic acids with respect to Watson-Crick base pairing and the potential of LNA containing oligonucleotides lies in their ability to mediate high affinity pairing with complementary RNA or DNA strands, with equal or often superior sequence specificity than their natural equivalent (4-7). Various applications and uses of LNA containing oligonucleotides have been reported and discussed in the literature. It has for example been shown that LNA containing oligonucleotides possess a gene silencing potential (7-9), that they are potential antisense drugs (10-12), and that they can be advantageously be used as probes in hybridization-based assays such as expression profiling, 44 DNA sequencing, and SNP genotyping (7, 13-19). It has 45 furthermore been shown that the introduction of LNA mono-46 mers into DNA oligonucleotides ensures substantial serum 47 stability, with low toxicity, which is a prerequisite for any 48 potential therapeutic use (20). 49 Numerous structural and thermodynamic studies have been 50 undertaken on LNA containing duplexes in order to try to 51 elucidate the factors at the origin of the observed increased 52 pairing selectivity and thermal stability. When LNA are incor-53 porated into DNA or RNA:DNA duplexes they maintain a right-54 handed helix conformation with all the bases in the anti 55 conformation. NMR studies however highlight that the incor-56 poration of LNA monomers into DNA duplexes induces the 57 local acquisition of A-type helix characteristics (21-26), with an 58 increased N-type contribution to the sugar conformation of the 59 DNA base-pairs adjacent to the incorporated LNA(23). The 60 incorporation of LNA nucleotides into the DNA strand of a 61 DNA:RNA hybrid duplex also leads to an increase in the 62 deoxyribose N-type conformation (22, 26). When a full LNA 63 strand is opposed to its cDNA strand the deoxyribose pucker 64 pattern is similar to the one observed in RNA:DNA (24). These 65 experimentally observed changes have been reproduced by 66 Molecular Dynamic simulations (27, 28). 67 The stability of LNA-containing duplexes is generally evalu-68 ated via thermal denaturation experiments. The observed increase 69 in the melting temperatures (T m) of LNA containing duplexes 70 relative to their native reference duplexes, range between þ1 and † G.B. thanks the Belgian "Fonds de la Recherche Scientifique-FNRS" for a postdoctoral fellowship. M.B. thanks the Belgian "Fonds pour la formation a la Recherche dans l'Industrie et dans l'Agriculture" for a Ph.D. grant.
Impact of the C1‘ Configuration of Abasic Sites on DNA Duplex Structure † , ‡
Biochemistry, 2004
Naturally occurring abasic sites in DNA exist as an equilibrium mixture of the aldehyde, the hydrated aldehyde, and the hemiacetal forms (dominant). The influence of the configuration of the C1′ hydroxyl group of the hemiacetal form on duplex structure and abasic site repair has been examined using novel carbocyclic analogues. Both the Rand -forms of this novel abasic site were introduced into oligomeric DNA using the standard DMT-phosphoramidite approach in an automated solid-phase synthesizer. Solution structures of the d(CGTACXCATGC)‚d(GCATGAGTACG) duplex (where X is the Ror -anomer of the carbocyclic abasic site analogue) were determined by NMR spectroscopy and restrained molecular dynamics simulations. The structures were only minimally perturbed by the presence of either anomer of the abasic site. All residues adopted an anti conformation, and Watson-Crick alignments were observed on all base pairs of the duplexes. At the lesion site, the abasic residues and their partner adenines showed increased dynamic behavior but adopted intrahelical positions in the final refined structures. Incision of duplexes having the Ror -anomer of the carbacyclic abasic site by human AP endonuclease showed that the enzyme recognizes both configurations of the lesion and nicks the DNA backbone with similar efficiency. Our results challenge the suggestion that Ape1 is stereoselective and imply a plasticity at the active site of the enzyme for accommodating either anomer of the lesion.
Biopolymers, 2015
The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (T g) lesion on a family of deoxyoligonucleotide duplexes. T g damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The T g lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates T g-induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-T g isomer centered within the tridecanucleotide sequence 5 0-GCGTACXCATGCG-3 0 (X 5 T g or T) which is hybridized with the corresponding complementary sequence 5 0-CGCATGNGTACGC-3 0 (N 5 A, G, T, C) to generate families of T g-damaged (T g ÁN) and lesion-free (TÁN) duplexes. We demonstrate that the magnitude and nature of the T g destabilizing impact is dependent on counterbase identity (i.e., A $ G < T < C). The observation that a T g lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of T g ÁN duplexes parallel their respective lesion-free TÁN mismatch counterparts (i.e., G < T < C). Elucidation of T g-induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The T g-induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery.
We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (AG') and the melting behavior (AW) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as (i) predicting the stability of a probe-gene complex; (ii) selecting optimal conditions for a hybridization experiment; (iii) deciding on the minimum length of a probe; (iv) predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and (v) predicting the relative stabilities of local domains within a DNA duplex. It is well established that under a given set of solution conditions the relative stability of a DNA duplex structure depends on its base sequence (1-4). More specifically, the stability of a DNA duplex appears to depend primarily on the identity of the nearest-neighbor bases. Ten different nearest-neighbor interactions are possible in any Watson-Crick DNA duplex structure. These pairwise interactions are AA/TT; AT/TA; TA/AT; CA/GT; GT/CA; CT/GA; GA/CT; CG/GC; GC/CG; GG/CC. The overall stability and the melting behavior of any DNA duplex structure can be predicted from its primary sequence if one knows the relative stability (AG') and the temperature-dependent behavior (Al?, ACp°) of each DNA nearest-neighbor interaction (5, 6). Tinoco and coworkers already have demonstrated the power of this predictive ability with RNA molecules for which they and others have determined the appropriate thermodynamic data (7-11). Unfortunately, comparatively few corresponding studies on DNA oligomers have been performed so that the relevant thermodynamic data required to predict DNA structural stability are rather sparse. The seriousness of this deficiency is dramatized by the fact that investigators attempting to evaluate sequence-dependent structural preferences in DNA molecules have resorted to the use of the more available RNA thermodynamic data. This use of RNA data does not reflect a belief that DNA and RNA are thermody-namically equivalent but rather is born of necessity due to a lack of the relevant DNA thermodynamic data. In fact, available comparisons suggest that serious errors may be introduced by applying RNA data to the analysis of sequence dependent structural preferences in DNA molecules (10, 12-14). Consequently, a meaningful evaluation of sequence dependent DNA structural preferences requires a DNA data base. Several years ago, we initiated a program with the expressed objective of obtaining the required DNA thermody-namic data. To this end, we have employed differential scanning calorimetry (DSC) and UV spectroscopy to characterize thermally induced helix-to-coil transitions in specially designed and synthesized oligomeric and polymeric DNA molecules (5, 6, 15-23). By combining the results from these studies, we now are able to resolve and to assign thermodynamic profiles for all 10 DNA nearest-neighbor interactions. Furthermore, we can demonstrate that DNA duplex structures thermodynamically can be considered to be the sum of their nearest-neighbor pairwise interactions. Consequently, using our nearest-neighbor DNA thermody-namic library we now can calculate the stability and predict the melting behavior of any DNA double helix from its primary sequence. This predictive ability should prove valuable in a number of important biochemical applications, such as calculating the minimum length of a probe oligomer required to form a stable duplex with a target gene at a given hybridization temperature, estimating the melting temperature of a duplex structure formed between an oligomeric probe and its complementary gene segment, identifying potential sites of local melting within a polymer duplex by predicting the sequence-dependent melting temperatures of local DNA domains, predicting the influence of a specific transition or transversion on the stability and melting temperature of a DNA sequence, and calculating and comparing the stability of a DNA duplex in the B conformation with the stability of the same sequence in alternative conformational states (e.g., Z, B', etc.) once these non-B conformations are thermodynamically characterized. In this article, we report the complete thermodynamic characterization of all 10 nearest-neighbor interactions possible in a Watson-Crick DNA duplex structure. More significantly , we demonstrate how these data can be used to predict the stability and the melting behavior of any DNA duplex from knowledge of its primary sequence. MATERIALS AND METHODS DNA Polymers and Oligomers. The three trimer-repeat polymers were synthesized by Robert Ratliff and were the kind gift of Tom Jovin. The remaining six polymers were obtained from P-L Biochemicals. We synthesized 12 of the 19 oligomers using the standard phosphotriester method (24). Three of the sequences [d(CGCGCG); d(CGTACG); and d(ATGCAT)] were the kind gift of our colleague Roger Jones. Abbreviation: DSC, differential scanning calorimetry. 3746 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Heat Capacity Changes Associated with DNA Duplex Formation: Salt- and Sequence-Dependent Effects †
Biochemistry, 2006
Duplexes are the most fundamental elements of nucleic acid folding. Although it has become increasingly clear that duplex formation can be associated with a significant change in heat capacity (∆C p), this parameter is typically overlooked in thermodynamic studies of nucleic acid folding. Analogy to protein folding suggests that base stacking events coupled to duplex formation should give rise to a ∆C p due to the release of waters solvating aromatic surfaces of nucleotide bases. In previous work, we showed that the ∆C p observed by isothermal titration calorimetry (ITC) for RNA duplex formation depended on salt and sequence [
Thermodynamics of DNA: heat capacity changes on duplex unfolding
European Biophysics Journal
The heat capacity change, ΔCp, accompanying the folding/unfolding of macromolecules reflects their changing state of hydration. Thermal denaturation of the DNA duplex is characterized by an increase in ΔCp but of much lower magnitude than observed for proteins. To understand this difference, the changes in solvent accessible surface area (ΔASA) have been determined for unfolding the B-form DNA duplex into disordered single strands. These showed that the polar component represents ~ 55% of the total increase in ASA, in contrast to globular proteins of similar molecular weight for which the polar component is only about 1/3rd of the total. As the exposure of polar surface results in a decrease of ΔCp, this explains the much reduced heat capacity increase observed for DNA and emphasizes the enhanced role of polar interactions in maintaining duplex structure. Appreciation of a non-zero ΔCp for DNA has important consequences for the calculation of duplex melting temperatures (Tm). A modi...