Hydrogen bonding versus stacking stabilization by modified nucleobases incorporated in PNA·DNA duplexes

The effects of incorporation of the modified nucleobases, 2,6-diaminopurine (D) (substituting for adenine) and 7-chloro-1,8-naphthyridin-2-(1H)-one (bicyclic thymine, bT) (substituting for thymine), that stabilize PNA·DNA duplex formation by increasing hydrogen bonding and/or base pair stacking interactions have been studied by thermal denaturation in terms of thermodynamics. Although the stabilizing effect of the bT base (in contrast to that of D base) is abolished upon addition of dimethyl formamide, thereby indicating that the stabilization is predominantly due to hydrophobic stacking forces, duplex stabilization was found to be enthalpic for both nucleobases. Increased stabilization (although not fully linearly) was observed with increasing numbers of modified bases, and single base sequence discrimination was only slightly compromised, but showed significant dependence on the sequence context.     

Potential of mean force calculations of the stacking-unstacking process in single-stranded deoxyribodinucleoside monophosphates.

The free energy of the stacking-unstacking process of deoxyribodinucleoside monophosphates in aqueous solution has been investigated by potential of mean force calculations along a reaction coordinate, defined by the distance between the glycosidic nitrogen atoms of the bases. The stacking-unstacking process of a ribodinucleoside monophosphate was observed to be well characterized by this coordinate, which has the advantage that it allows for a dynamical backbone and flexible bases. All 16 naturally occurring DNA dimers composed of the adenine, cytosine, guanine, or thymine bases in both the 5' and the 3' positions were studied. From the free-energy profiles we observed the deepest minima for the stacked states of the purine-purine dimers, but good stacking was also observed for the purine-pyrimidine and pyrimidine-purine dimers. Substantial stacking ability was found for the dimers composed of a thymine base and a purine base and also for the deoxythymidylyl-3',5'-deoxythymidine dimer. Very poor stacking was observed for the dCpdC dimer. Conformational properties and solvent accessibility are discussed for the stacked and unstacked dimers. The potential of mean force profiles of the stacking-unstacking process for the DNA dimers are compared with the RNA dimers.     

Adsorption of Adenine and Thymine on Zeolites: FT-IR and EPR Spectroscopy and X-Ray Diffractometry and SEM Studies

The interactions of adenine and thymine with and adsorption on zeolites were studied using different techniques. There were two main findings. First, as shown by X-ray diffractometry, thymine increased the decomposition of the zeolites (Y, ZSM-5) while adenine prevented it. Second, zeolite Y adsorbed almost the same amount of adenine and thymine, thus both nucleic acid bases could be protected from hydrolysis and UV radiation and could be available for molecular evolution. The X-ray diffractometry and SEM showed that artificial seawater almost dissolved zeolite A. The adsorption of adenine on ZSM-5 zeolite was higher than that of thymine (Student-Newman-Keuls test-SNK p<0.05). Adenine was also more greatly adsorbed on ZSM-5 zeolite, when compared to other zeolites (SNK p<0.05). However the adsorption of thymine on different zeolites was not statistically different (SNK p>0.05). The adsorption of adenine and thymine on zeolites did not depend on pore size or Si/Al ratio and it was not explained only by electrostatic forces; rather van der Waals interactions should also be considered.     

Perturbation of hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations

Perturbation of the hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine...thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at O6 leads to destabilization of the adenine...thymine pair; divalent cations (Mg2+, Ca2+) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

Ab-initio quantum mechanical calculations of NMR chemical shifts in nucleic acids constituents. III. Chemical shift variations due to base stacking

Ab inito computations of the different contributions to chemical shift variations due to intra and interstrand stacking are reported for the GC, CG, AT and TA sequences of a B DNA helix. The results obtained for the non hydrogen atoms of the GC stacks show that the chemical shift variations are mainly due to the polarization contribution, the term which decreases slowly with the intermolecular distance. Because of the weaker polarity of adenine and thymine the geometric and polarization contributions are of closer absolute magnitude for the non hydrogen atoms of the intrastrand stacks but the polarization term is the determining contribution in the corresponding interstrand stacks. For the protons which undergo smaller shifts due to the polarization (or electric field effects) the role of the geometric contribution is more important and is even the leading one for the hydrogens of cytosine and thymine in the case of intrastrand stacking. The charge transfer plus exchange term has a non negligeable value for a limited number of cases corresponding to the shortest intermolecular interatomic distances. These results are discussed in relation with the qualitative differences observed between the proton and carbon spectra of dinucleotides and B-DNA duplexes.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: specific reactivity with short A-tracts

We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G). The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene. The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent. In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity. In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts. Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine. Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C. However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost. Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures. The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA. This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts. The relevance of these results to current models of A-tract structure and DNA bending is discussed. Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.     

Perturbation of Hydrogen Bonds in the Adenine…Thymine Base Pair by Na+, Mg2+, Ca2+ and NH4 + Cations

Abstract

Perturbation of the hydrogen bonds in the adenine…thymine base pair by Na⁺, Mg²⁺, Ca²⁺ and NH₄ ⁺ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine…thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at 06 leads to destabilization of the adenine…thy mine pair; divalent cations (Mg²⁺, Ca²⁺) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

Luminescence of stable stacking aggregates of adenine and uracil in water

Luminescence and excitation spectra of the highly luminescent stacking dimers of adenine and uracil in water solutions are studied. By the luminescence excitation spectra, it is shown that the stacking aggregates of adenine and uracil are formed with participation of rare forms of monomers: adenine N7H tautomers, and uracil in uncommon hydration states, e.g. lacking H-bonds with water. The temperature dependence of luminescence intensity of monomers and stacking dimers of uracil shows that stacking dimers do not dissociate even at 85°C, just as described earlier for adenine and adenosine. Stable stacking aggregates of nucleic bases are the likely precursors of RNA molecules in chemical evolution. This hypothesis is supported by new data on their heat stability.     

Luminescence of stable stacking aggregates of adenine and uracil in water

Luminescence and excitation spectra of the highly luminescent stacking dimers of adenine and uracil in water solutions are studied. By the luminescence excitation spectra method it is shown that the stacking aggregates of adenine and uracil are formed with participation of rare forms of monomer molecules: N7H tautomers of adenine and the uracil molecules in rare forms of hydratation, for example molecules without H-bonds with water. The study of temperature dependence of luminescence intensity of monomers and stacking dimers of uracil has shown that stacking dimers do not dissociate even at 85 degrees C similarly as described earlier for adenine and adenosine. Stable stacking aggregates of nucleic bases are most likely to be the precursors of RNA molecules in chemical evolution. This hypothesis is supported by new data on their stability.     

Propeller-twisted adenine.thymine and guanine.cytosine base pairs tend to buckle and stagger in opposite directions.

Base pairs are propeller-twisted, buckled and staggered in DNA fragment crystals. These deformations were analyzed with isolated Watson-Crick base pairs using empirical potentials and buckle was found to almost linearly correlate with propeller. Interestingly, the thymine.adenine pair favours negative buckling for propellers mostly observed in DNA crystals while positive buckling is preferred by the cytosine.guanine pair. The propeller also induces opposite staggers in the adenine.thymine and guanine.cytosine base pairs.     

Denaturation map of the ColE1-Km plasmid pCR11.

The denaturation map of EcoRI-digested pCR11, a ColE1-Km plasmid, is described. The 2.0 kilobase ColE1-derived segment contains an adenine+thymine rich site in the colicin immunity gene region. In the 7.2 kilobase kanamycin resistance region, the transposon Tn903 consists of an adenine+thymine rich 0.98 kilobase kan gene region flanked by a guanine+cytosine rich 1.09 kilobase inverted duplication.     

Biased distribution of adenine and thymine in gene nucleotide sequences

We analyzed occurrences of bases in 20,352 introns, exons of 25,574 protein-coding genes, and among the three codon positions in the protein-coding sequences. The nucleotide sequences originated from the whole spectrum of organisms from bacteria to primates. The analysis revealed the following: (1) In most exons, adenine dominates over thymine. In other words, adenine and thymine are distributed in an asymmetric way between the exon and the complementary strand, and the coding sequence is mostly located in the adenine-rich strand. (2) Thymine dominates over adenine not only in the strand complementary to the exon but also in introns. (3) A general bias is further revealed in the distribution of adenine and thymine among the three codon positions in the exons, where adenine dominates over thymine in the second and mainly the first codon position while the reverse holds in the third codon position. The product (A₁/T₁) × (A₂/T₂) × (T₃/A₃) is smaller than one in only a few analyzed genes. Correspondence to: J. Kypr     

1,2-DISUBSTITUTED CYCLOHEXANE CARBOCYCLIC ANALOGUES OF NUCLEOSIDES

Several compounds of a new series of cyclohexane-based 1,2-disubstituted carbonucleoside analogues, were synthesized. The adenine and uridine derivatives, were prepared by construction of the heterocyclic base on the primary amino group of 2-aminocyclohexylmethanol, and the thymine derivative by condensation of 2-hydroxycyclohexylmethanol with thymine using the Mitsunobu reaction.     

1,2-disubstituted cyclohexane carbocyclic analogues of nucleosides

Several compounds of a new series of cyclohexane-based 1,2-disubstituted carbonucleoside analogues, were synthesized. The adenine and uridine derivatives, were prepared by construction of the heterocyclic base on the primary amino group of 2-aminocyclohexylmethanol, and the thymine derivative by condensation of 2-hydroxycyclohexylmethanol with thymine using the Mitsunobu reaction.     

Photochemical reactions on the synthetic polymers containing thymine bases

Photodimerizations of N-2-isobutyloxyethyl thymine (T-M), bis[2-(5-methyl-1-pyrimidinyl)ethyl]glutarate (T-T), poly-N-2-methacryloyloxethyl thymine (P-MAOT) and poly-N-2-acryloyloxyethyl thymine (P-AOT) were studied in dimethylformamide solution. Quantum efficiencies of intramolecular photodimerizations were determined to be 0.0012 for T-T, 0.0084 for P-MAOT and 0.010 for P-AOT. In the case of T-M, however, intermolecular photodimerization did not occur under the reaction condition used. Quenching studies by using isoprene suggest that the photodimerization of T-T occurs predominantly through an excited triplet state, while that of P-MAOT and P-AOT occur through both singlet and triplet states. For the effect of adding model compounds containing adenine bases on this reaction, adenine derivatives acts as an inhibitor against this reaction by quenching the excited singlet state of thymine. The photodimers of T-T, P-MAOT and P-AOT were concluded to be two syn-fused cyclobutane- type dimers (cis-syn and trans-syn).     

Crystallographic studies on damaged DNAs. II. N(6)-methoxyadenine can present two alternate faces for Watson-Crick base-pairing, leading to pyrimidine transition mutagenesis

In a previous paper, 2'-deoxy-N(6)-methoxyadenosine (mo(6)A) was shown to form a mismatch base-pair with 2'-deoxycytidine with a Watson-Crick-type geometry. To fully understand the structural basis of genetic mutations with damaged DNA, it is necessary to examine whether the methoxylated adenine residue still has the ability to form the regular Watson-Crick pairing with a thymine residue. Therefore, a DNA dodecamer with the sequence d(CGCGmo(6)AATTCGCG) has been synthesized and its crystal structure determined. The methoxylation has no significant effect on the overall DNA conformation, which is that of a standard B-form duplex. The methoxylated adenine moieties adopt the amino tautomer with an anti conformation around the C(6)-N(6) bond to the N(1) atom, and they form a Watson-Crick base-pair with thymine residues on the opposite strand, similar to an unmodified adenine residue. It is concluded that methoxylated adenine can present two alternate faces for base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.     

DNA synthesis in relation to a varying vitamin B12 content in Propionibacterium shermanii cells

The content of DNA in Propionibacterium cells is nearly twice as low at vitamin B12 deficiency comparing with normal cells. The rate of labeled adenine incorporation into the DNA depends on vitamin B12 content in the cells. The addition of adenosylcobalamin and thymine to the medium makes DNA content rise in the cells of the B12-deficient culture. The addition of thymine to a suspension of B12-deficient cells accelerates the incorporation of labelled adenine into DNA. The authors discuss the phenomenon of bacterial "unbalanced division" and the control of replication by changing the level of DNA precursors.