A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

Intercalation of daunomycin into stacked DNA base pairs. DFT study of an anticancer drug

We have computationally studied the intercalation of the antitumor drug daunomycin into six stacks of Watson-Crick DNA base pairs (i.e., AT-AT, AT-TA, GC-AT, CG-TA, GC-GC, GC-CG) using density functional theory (DFT). The proton affinity of the DNA intercalator daunomycin in water was computed to be 159.2 kcal/mol at BP86/TZ2P, which is in line with the experimental observation that daunomycin is protonated under physiological conditions. The intercalation interaction of protonated daunomycin with two stacked DNA base pairs was studied through a hybrid approach in which intercalation is treated at LDA/TZP while the molecular structure of daunomycin and hydrogen-bonded Watson-Crick pairs is computed at BP86/TZ2P. We find that the affinity of the drug for the six considered base pair dimers decreases in the order AT-AT > AT-TA > GC-AT > GC-TA > GC-CG > GC-GC, in excellent agreement with experimental data on the thermodynamics of the interaction between daunomycin and synthetic polynucleotides in aqueous solution. Our analyses show that the overall stability of the intercalation complexes comes mainly from pi-pi stacking but an important contribution to the computed and experimentally observed sequence specificity comes from hydrogen bonding between daunomycin and hetero atoms in the minor groove of AT base pairs.     

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis of novel base pairs and their enzymatic incorporation into DNA.

A novel base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and the counter part, pyridin-2-one (denoted y) were designed. The bulky 6-dimethylamino group of x is expected to eliminate base pairing with all natural bases. The phosphoramidite of x for DNA templates and the 2'-deoxyribonucleoside triphosphate of y (dyTP) for a substrate were synthesized, and the selectivity of the enzymatic incorporation of dyTP opposite x in the templates was examined. dyTP was preferentially incorporated opposite x than canonical dNTPs by Klenow fragment of Escherichia coli DNA polymerase I. While dyTP was also incorporated opposite A and G, the misincorporation was suppressed in the presence of dTTP and dCTP, respectively.     

Circular dichroism measurements show that C.C+ base pairs can coexist with A.T base pairs between antiparallel strands of an oligodeoxynucleotide double-helix.

We have studied the coil-to-helix transition of the DNA oligomer d(C4A4T4C4), using circular dichroism measurements to monitor the formation of A.T base pairs within the central self-complementary A4T4 region and the formation of protonated C.C+ base pairs at the ends of the oligomer. We found that both A.T and C.C+ base pairs formed in a coordinated fashion as the temperature and pH were lowered. The CD data of the helix form of the oligomer were consistent with the presence of paired oligomers, but not with hairpin loops. The pKa for formation of C.C+ base pairs between the C4 ends of the oligomer was higher than the pKa for formation of C.C+ base pairs in d(C8), indicating that the formation of C.C+ base pairs in the oligomer was influenced by the presence of a paired A4T4 region. We conclude that A.T and C.C+ base pairs coexist in the self-complex of the oligomer and, therefore, that C.C+ base pairs can form between antiparallel DNA strands.     

Thermodynamic properties of the specific binding between Ag+ ions and C:C mismatched base pairs in duplex DNA

Metal-mediated base pairs formed by the interaction between metal ions and artificial bases in oligonucleotides have been developed for potential applications in nanotechnology. We recently found that a natural C:C mismatched base pair bound to an Ag(+) ion to generate a novel metal-mediated base pair in duplex DNA. Preparation of the novel C-Ag-C base pair involving natural bases is more convenient than that of metal-mediated base pairs involving artificial bases because time-consuming base synthesis is not required. Here, we examined the thermodynamic properties of the binding between the Ag(+) ion and each of single and double C:C mismatched base pair in duplex DNA by isothermal titration calorimetry. The Ag(+) ion specifically bound to the C:C mismatched base pair at a 1:1 molar ratio with 10(6) M(-1) binding constant, which was significantly larger than those for nonspecific metal ion-DNA interactions. The specific binding between the Ag(+) ion and the single C:C mismatched base pair was mainly driven by the positive dehydration entropy change and the negative binding enthalpy change. In the interaction between the Ag(+) ion and each of the consecutive and interrupted double C:C mismatched base pairs, stoichiometric binding at a 1:1 molar ratio was achieved in each step of the first and second Ag(+) binding. The binding affinity for the second Ag(+) binding was similar to that for the first Ag(+) binding. Stoichiometric binding without interference and negative cooperativity may be favorable for aligning multiple Ag(+) ions in duplex DNA for applications of the metal-mediated base pairs in nanotechnology.     

Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.     

Dynamics of mismatched base pairs in DNA

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.     

A new model for DNA containing A.T and I.C base pairs.

DNA polymers containing exclusively A.T or I.C base pairs frequently exhibit D- or E-type X-ray diffraction patterns when dried. The distribution of intensities in fiber patterns appears to demand helical structures with 7 and 7.5 bp/turn, respectively, but it is not stereochemically possible to wind a right-handed antiparallel B-family helix this tightly. It is a simple matter, however, to build a left-handed helix with 7-7.5 bp/turn by incorporating Hoogsteen pairing into a Z helix framework. X-ray intensities calculated from this novel left-handed Hoogsteen model provide as reasonable a fit to the D-DNA diffraction pattern as do intensities calculated from previously proposed right-handed 8-fold models.     

Molecular recognition of DNA base pairs by the formamido/pyrrole and formamido/imidazole pairings in stacked polyamides

Polyamides containing an N-terminal formamido (f) group bind to the minor groove of DNA as staggered, antiparallel dimers in a sequence-specific manner. The formamido group increases the affinity and binding site size, and it promotes the molecules to stack in a staggered fashion thereby pairing itself with either a pyrrole (Py) or an imidazole (Im). There has not been a systematic study on the DNA recognition properties of the f/Py and f/Im terminal pairings. These pairings were analyzed here in the context of f-ImPyPy, f-ImPyIm, f-PyPyPy and f-PyPyIm, which contain the central pairing modes, –ImPy– and –PyPy–. The specificity of these triamides towards symmetrical recognition sites allowed for the f/Py and f/Im terminal pairings to be directly compared by SPR, CD and ΔT_M experiments. The f/Py pairing, when placed next to the –ImPy– or –PyPy– central pairings, prefers A/T and T/A base pairs to G/C base pairs, suggesting that f/Py has similar DNA recognition specificity to Py/Py. With –ImPy– central pairings, f/Im prefers C/G base pairs (>10 times) to the other Watson–Crick base pairs; therefore, f/Im behaves like the Py/Im pair. However, the f/Im pairing is not selective for the C/G base pair when placed next to the –PyPy– central pairings.     

Spin-orbit couplings in the DNA base pairs

The radiative lifetimes of the phosphorescent states of the adenine.thymine (A.T) and guanine.cytosine (G.C) base pairs were calculated on the basis of the singlet-triplet transition probability induced by spin-orbit couplings. The calculated radiative lifetimes averaged over the triplet sublevels of spin state were in the order of G.C less than A.T and in good correlation with those of the composite bases. On the whole the results suggested an important role for thymine triplet having a relatively long lifetime during the course of the triplet localization in DNA, in agreement with the experimental observation that the concentration of triplet is remarkably enhanced with increase in A+T content.     

An NMR structural study of deaminated base pairs in DNA.

The structurally aberrant base pairs TG, UG and TI may occur in DNA as a consequence of deamination of 5-methylcytosine, cytosine and adenine respectively. Results of NMR spectroscopic studies are reported here for these deaminated base pairs in a model seven base pair long oligonucleotide duplex. We find that in all three cases, the DNA helix is a normal B form and both mispaired bases are intrahelical and hydrogen bonded with one another in a wobble geometry. Similarly, in all three cases, all sugars are found to be normal C2' endo in conformation. Symmetric structural perturbations are observed in the helix twist on the 3' side of the mispaired pyrimidine and on the 5' side of the mispaired purine. In all three cases, the amino group of the G residue on the 3' side of the mispaired pyrimidine shows hindered rotation. Although less thermodynamically stable than helices containing only Watson-Crick base pairs, these helices melt normally from the ends and not from the mispair outwards.     

Excited states of the DNA base pairs

The results of calculations of the first π-electronic states of the DNA base pairs with the SCF-MO-LCAO method both without taking into account configuration interaction and taking into account all the singly excited configurations are presented. The first excited singlet state and the first triplet state of both pairs are shown to be, independently of an approximation, the states where the excitation is localized on one of the bases.     

Do Hoogsteen base pairs occur in DNA?

The importance of the Watson-Crick complementary base-pairing scheme has rather overshadowed alternative types of base pairs in DNA. One of these alternative base pairings, which is known as Hoogsteen pairing, is now receiving attention. Its presence in crystals of oligonucleotides bound to some antibiotics, and its possible existence in solution (and within long DNA fragments) remains to be unambiguously estimated. However, variability in DNA conformation appears to play an important biological role, and thus we should consider the presence of Hoogsteen base pairs as an interesting factor in inducing such changes.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Recognition of base pairs by polar peptides in double stranded DNA.

The resonance Raman spectrum of native DNA has been obtained using excitation at 257 nm. In a first part, the spectral lines are assigned to the different nucleotide bases which provide the resonance effect. In a second part, the interactions of DNA with basic peptides (Arginine Methylester, Lysine Methylester, Arginyl-Arginine) are investigated using excitation at 300 nm and 257 nm, which give complementary information about the DNA. Both Arginine Methylester and Arginyl-arginine recognize the A-T base pairs, the first one in the large groove, the second one in the narrow groove of DNA. The DNA-Lysine Methylester interaction is very likely not specific but can take place in the large groove of DNA.     

Vibrational fluctuations of hydrogen bonds in a DNA double helix with nonuniform base pairs.

The Green's function technique is applied to a study of breathing modes in a DNA double helix which contains a region of different base pairs from the rest of the double helix. The calculation is performed on a G-C helix in the B conformation with four consecutive base pairs replaced by A-T. The average stretch in hydrogen bonds is found amplified around the A-T base pair region compared with that of poly(dG)-poly(dC). This is likely related to the A-T regions lower stability against hydrogen bond melting. The A-T region may be considered to be the initiation site for melting in such a helix.     

The intrinsic structure and stability of out-of-alternation base pairs in Z-DNA.

Alternating pyrimidine-purine sequences typically form Z-DNA, with the pyrimidines in the anti and purines in the syn conformations. The observation that dC and dT nucleotides can also adopt the syn conformation (i.e. the nucleotides are out-of-alternation) extends the range of sequences that can convert to this left-handed form of DNA. Here, we study the effects of placing two adjacent d(G*C) base pairs as opposed to a single d(G*C) base pair or two d(A*T) base pairs out-of-alternation by comparing the structure of d(m5CGGCm5CG)2with the previously published structures of d(m5CGGGm5CG)*d(m5CGCCm5CG) and d(m5CGATm5CG)2. A high buckle and loss of stacking interactions are observed as intrinsic properties of the out-of-alternation base pairs regardless of sequence and the context of the dinucleotide. From solution titrations, we find that the destabilizing effect of out-of-alternation d(G*C) base pairs are identical whether these base pairs are adjacent or isolated. We can therefore conclude that it is these intrinsic distortions in the structure of the base pairs and not neighboring effects that account for the inability of out-of-alternation base pairs to adopt the left-handed Z conformation.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.