Chemical consequences of incorporation of 5-fluorouracil into DNA as studied by NMR

The cytotoxic analogue of thymine, 5-fluorouracil (Uf), is known to be incorporated into DNA in biological systems. This abnormal base has been synthetically incorporated into short DNA oligomers. The ionization of the N-3 proton of this base within DNA oligomers was measured by observation of the 19F chemical shift at varying pH values. The pKa values for the Uf ring of dTpdUfpdT and dApdUfpdA were determined to be 7.84 and 7.9, respectively. The self-complementary 12-mers d(G-C-G-C-A-A-T-Uf-G-C-G-C) and d(C-G-A-T-Uf-A-T-A-A-T-C-G) were synthesized, and 1H NMR was used to compare the helix dynamics and stability of the interstrand imino proton hydrogen bonds with those of the 12-mers d(G-C-G-C-A-A-T-T-G-C-G-C) and d(C-G-A-T-T-A-T-A-A-T-C-G). The N-3 hydrogen bond of the A-Uf base pair was less stable than the corresponding hydrogen bond in A-T base pairs in the same helix, and the A-Uf base pair was less stable than the A-T base pair in the analogous position of the control helix. The observed temperature-dependent dynamics and NMR melting temperatures of the control and dUf-containing oligomers were similar.     

A new calculation on spectrum of direct DNA damage induced by low-energy electrons

In this work, direct DNA damage induced by low-energy electrons (<5 keV) is simulated using Monte Carlo methods, and the resulting yield of various strand breaks and base damages in cellular environment is presented. The simulation is based on a new inelastic cross section for the production of electron track structure in liquid water, and on ionization cross sections of DNA bases to generate base radical. Especially, a systematic approach of simulating detailed base damage is suggested. This approach includes improvement of a volume model of DNA, generation of the DNA base sequence, conversion of ionization events in liquid water at hit site to the ionization interaction of electrons with DNA bases and development of an algorithm to convert a base radical to a damage. The results obtained in terms of strand breaks are compared with those of experiments and other theoretical calculations, and good agreement was obtained. The yield of detailed base damages and clustered DNA damages caused by the combination of various strand breaks and base damages is presented, and the corresponding distribution characteristics are analyzed. The influence of the relative content of base pairs A-T and G-C in a DNA segment on the yield of both strand breaks and base damages is also explored. The present work provides fundamental information on DNA damage and represents the first effort toward the goal of obtaining the spectrum of clustered DNA damage including detailed base damages, for the mechanistic interpretation and prediction of radiation effects.     

Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase

The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.     

Specificity of mutagenesis by 4-aminobiphenyl. A possible role for N-(deoxyadenosin-8-yl)-4-aminobiphenyl as a premutational lesion

Mutagenesis by N-acetoxy-N-trifluoroacetyl-4-aminobiphenyl, a reactive form of the human bladder carcinogen 4-aminobiphenyl (ABP), was studied in Escherichia coli virus M13mp10. N-acetoxy-N-trifluoroacetyl-4-ABP-treated DNA containing 140 lesions/duplex genome, when introduced into excision repair-competent cells induced for SOS mutagenic processing, resulted in a 40-fold increase in mutation frequency over background in the lacZ alpha gene fragment. DNA sequence changes were determined for 20 independent mutants. G-C base pairs were the major targets for base pair substitution mutations, although significant mutagenic activity was also observed at certain A-T base pairs. Deletion and frameshift mutations also were found in this sample. The salient feature of this partial "mutational spectrum" was a hotspot that occurred at position 6357 (amino acid 30 of the beta-galactosidase fragment encoded by M13mp10); this A-T to T-A transversion appeared in 6 of the 20 mutants. The property of ABP to mutate A-T base pairs was consistent with the result that N-hydroxy-ABP reverted Salmonella typhimurium strain TA104, which is presumed to revert primarily due to mutations at these sites. The ability of the major carcinogen-DNA adduct formed by ABP in vivo and in vitro, N-(deoxyguanosin-8-yl)-4-aminobiphenyl, to cause base pair substitution mutations was also investigated. This adduct was positioned specifically in the minus strand at position 6270 in duplex M13mp10 DNA. In the presence of the mutagenesis-enhancing plasmid pGW16 and UV induction of SOS mutagenic processing, it was shown that fewer than 0.02% of the adducts resulted in transition or transversion mutations following transfection of DNA into excision-repair competent cells. Similar results were obtained in uvrA and uvrC backgrounds. Although the major adduct did not cause base substitution mutations under these experimental conditions, the contribution of this lesion to the entire spectrum of mutations in the lacZ alpha fragment seems likely.     

Studies on tautomeric forms of Guanine-Cytosine base pairs of nucleic acids and their interactions with water molecules

The relative stabilities of Guanine-Cytosine (G-C) DNA bare base pairs, its tautomeric forms and microhydrated base pairs are theoretically investigated with a focus on the keto-enol tautomerism as well as on the cis-trans isomerism using ab initio and density functional theory methods. The stabilities of the G-C bare base pairs, its tautomeric forms and microhydrated base pairs were affected by various factors including keto-enol tautomerization, cis-trans enol isomerization, and steric hindrance between the base pair and water molecules. The Atoms in Molecules theory (AIM) is employed to investigate H-bonding patterns both in bare and microhydrated base pairs. From the above topological results, an excellent linear correlation is shown between electron density [rho(r)], and its Laplacian [V2rho(r)] at the bond critical points. NBO analysis has been carried out to study the charge transfer between proton acceptor to the antibonding orbital of the X-H bond both in bare and microhydrated base pairs.     

Post Hartree-Fock studies of the canonical Watson-Crick DNA base pairs: molecular structure and the nature of stability

Gas-phase gradient optimization was carried out on the canonical Watson-Crick DNA base pairs using the second-order M?ller-Plesset perturbation method at the 6-31G(d) and 6-31G(d,p) basis sets. It is detected that full geometry optimization at the MP2 level leads to an intrinsically nonplanar propeller-twisted and buckled geometry of G-C and A-T base pairs; while HF and DFT methods predict perfect planar or almost planar geometry of the base pairs. Supposedly the nonplanarity of the pairs is caused by pyramidalization of the amino nitrogen atoms, which is underestimated by the HF and DFT methods. This justifies the importance of geometry optimization at the MP2 level for obtaining reliable prediction of the charge distribution, molecular dipole moments and geometrical structure of the base pairs. The Morokuma-Kitaura and the Reduced Variational Space methods of the decomposition for molecular HF interaction energies were used for investigation of the hydrogen bonding in the Watson-Crick base pairs. It is shown that the HF stability of the hydrogen-bonded DNA base pairs originates mainly from electrostatic interactions. At the same time, the calculated magnitude of the second order intramolecular correlation correction to the Coulomb energy showed that electron correlation reduces the contribution of the electrostatic term to the attractive interaction for the A-T and G-C base pairs. Polarization, charge transfer and dispersion interactions also make considerable contribution to the attraction energy of bases.     

Structural basis for uracil recognition by archaeal family B DNA polymerases

Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-C-->A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.     

Formation of strand breaks and interstrand cross-links in DNA by methylglyoxal

Methylglyoxal (MG), a dietary mutagen, is present in various frequently consumed beverages and foods and in cigarette smoke. A combination of S1 nuclease hydrolysis and alkaline unwinding assay was used to demonstrate the formation of single-strand breaks and interstrand cross-links in DNA upon treatment with MG. Calf thymus DNA, when treated with increasing concentrations of MG, showed an increasing degree of S1 nuclease hydrolysis. It also showed the formation of an increasing number of strand breaks per molecule as determined by an alkaline unwinding assay. Incubation of DNA with relatively higher concentrations of methylglyoxal or prolonged treatment gave increased thermal melting temperatures and an enhanced rate of reannealing after thermal denaturation. These results indicated the formation of interstrand cross-links. Upon treatment with MG, A-T base pair depleted DNA showed a reduced number of single-strand break formation. It also showed a significantly lower decrease in Tm as compared with MG-treated normal DNA. These results showed that under the conditions used, MG primarily reacts with A-T base pairs in duplex DNA.     

Post Hartree-Fock Studies of the Canonical Watson-Crick DNA Base Pairs: Molecular Structure and the Nature of Stability

Abstract

Gas-phase gradient optimization was carried out on the canonical Watson-Crick DNA base pairs using the second-order Møller-Plesset perturbation method at the 6–31G(d) and 6- 31G(d,p) basis sets. It is detected that full geometry optimization at the MP2 level leads to an intrinsically nonplanar propeller-twisted and buckled geometry of G-C and A-T base pairs; while HF and DFT methods predict perfect planar or almost planar geometry of the base pairs. Supposedly the nonplanarity of the pairs is caused by pyramidalization of the amino nitrogen atoms, which is underestimated by the HF and DFT methods. This justifies the importance of geometry optimization at the MP2 level for obtaining reliable prediction of the charge distribution, molecular dipole moments and geometrical structure of the base pairs. The Morokuma-Kitaura and the Reduced Variational Space methods of the decomposition for molecular HF interaction energies were used for investigation of the hydrogen bonding in the Watson-Crick base pairs. It is shown that the HF stability of the hydrogen-bonded DNA base pairs originates mainly from electrostatic interactions. At the same time, the calculated magnitude of the second order intramolecular correlation correction to the Coulomb energy showed that electron correlation reduces the contribution of the electrostatic term to the attractive interaction for the A-T and G-C base pairs. Polarization, charge transfer and dispersion interactions also make considerable contribution to the attraction energy of bases.     

1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove

A symmetrical cyanine dye was previously shown to bind as a cofacial dimer to alternating A-T sequences of duplex DNA. Indirect evidence suggested that dimerization of the dye occurred in the minor groove. 1H NMR experiments reported here verify this model based on broadening and shifting of signals due to protons on carbon 2 of adenine and imino protons at the central five A-T pairs of the 11 base pair duplex: 5'-GCGTATATGCG-3'/3'-CGCATATACGC-5'. This binding mode is similar to that of distamycin A, even though the dye lacks the hydrogen-bonding groups used by distamycin for sequence-specific recognition. Surprisingly, the third base pair (G-C) was also implicated in the binding site. UV-vis experiments were used to compare the extent of dimerization of the dye for 11 different sequence variants. These experiments verified the importance of a G-C pair at the third position: replacing this pair with A-T suppressed dimerization. These results indicate that the dye binding site spans six base pairs: 5'-GTATAT-3'. The initial G-C pair seems to be important for widening the minor groove rather than for making important contacts with the dye molecules since inverting its orientation to C-G or replacing it with I-C still led to favorable dimerization of the dye.     

Evaluation of intrinsic spectroscopic properties of chromophore assemblies by shielding with cyclohexyl base pairs within a DNA duplex

Here, we investigated spectroscopic behaviors of tetramethylrhodamine (TMR) homo- and hetero-dimers within DNA duplex. In order to shield the chromophores from natural base pairs, we used cyclohexyl base pairs as ‘insulators’; these pairs were inserted between the chromophores and nucleobases. When a single TMR moiety was sandwiched between cyclohexyl base pairs, the emission intensity increased by fivefold relative to a TMR between natural base pairs, because electron transfer from nucleobases was suppressed. Next, we inserted two TMRs between the cyclohexyl base pairs and found that they facilitated H-dimer formation of TMR; a distinct hypsochromic shift was induced only when cyclohexyl base pairs were inserted. We further examined quenching behavior of a TMR paired with a quencher dye between cyclohexyl base pairs. Interestingly, fluorescence from TMR was quenched by nitro methyl red more efficiently in the presence of cyclohexyl base pairs than in their absence. This suggests that neighboring natural base pairs disturbed electron or hole transfer between the fluorophore and the quencher. The cyclohexyl base pairs shielded the chromophore pair from the natural base pairs and allowed intrinsic electron transfer.     

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.     

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

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

Study of the hydrogen bond in different orientations of adenine-thymine base pairs: An <Emphasis Type="Italic">ab initio</Emphasis> study

In order to gain deeper insight into structure, charge distribution, and energies of A-T base pairs, we have performed quantum chemical ab initio and density functional calculations at the HF (Hartree-Fock) and B3LYP levels with 3-21G*, 6-31G*, 6-31G**, and 6-31++G** basis sets. The calculated donor-acceptor atom distances in the Watson-Crick A-T base pair are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. In addition, for further correction of interaction energies between adenine and thymine, the basis set superposition error (BSSE) associated with the hydrogen bond energy has been computed via the counterpoise method using the individual bases as fragments. In the Watson-Crick A-T base pair there is a good agreement between theory and experimental results. The distances for (N2...H23-N19), (N8-H13...O24), and (C1...O18) are 2.84, 2.94, and 3.63 A, respectively, at B3LYP/6-31G** level, which is in good agreement with experimental results (2.82, 2.98, and 3.52 A). Interaction energy of the Watson-Crick A-T base pair is -13.90 and -10.24 kcal/mol at B3LYP/6-31G** and HF/6-31G** levels, respectively. The interaction energy of model (9) A-T base pair is larger than others, -18.28 and -17.26 kcal/mol, and for model (2) is the smallest value, -13.53 and -13.03 kcal/mol, at B3LYP/6-31G** and B3LYP/6-31++G** levels, respectively. The computed B3LYP/6-31G** bond enthalpies for Watson-Crick A-T pairs of -14.4 kcal/mol agree well with the experimental results of -12.1 kcal/mol deviating by as little as -2.3 kcal/mol. The BSSE of some cases is large (9.85 kcal/mol) and some is quite small (0.6 kcal/mol).     

Electron transfer chemistry between DNA and DNA-binding tripeptides

A DNA system consisting of pyrene-modified oligonucleotides and nitrobenzoate (Nb)-modified DNA-binding tripeptides has been applied to study electron-transfer processes through the DNA-peptide interface. 5-(Pyren-1-yl)-2'-deoxyuridine (Py-dU) has been used as the photoinducible charge generator. Upon excitation at 350 nm, a pyrene-like excited state (Py-dU) is formed which undergoes an electron transfer yielding the charge-separated state which is the contact ion pair Py(*)(+)-dU(*)(-). The subsequent electron shift from dU(*)(-) into the base stack competes with charge recombination and can be probed chemically by trapping the electron at the 5-bromo-2'-deoxyuridine (Br-dU) group leading to strand cleavage which can be quantified by HPLC analysis. Several Nb-modified DNA-binding tripeptides influence these DNA-mediated electron-transfer processes as shown by fluorescence spectroscopy experiments. Fluorescence quenching can occur primarily through a reductive electron-transfer process in which the Nb group traps the electron thermodynamically from the contact ion pair Py(*)(+)-dU(*)(-). Moreover, our results indicate that, once the negative charge has been trapped on the peptide, oxidative processes from Py(*)(+) take place resulting in an enhanced and nonspecific strand degradation of the Py-dU-modified duplexes. The latter type of strand cleavage can be inhibited by the presence of tryptophane or tyrosine as part of the peptides. Most remarkably, DNA-binding tripeptides, which bear both the Nb and the tryptophan/tyrosine moiety, are able to trap both the negative and the positive charge from the contact ion pair Py(*)(+)-dU(*)(-).     

Counterstain-enhanced chromosome banding

Summary Chromosome staining, in which at least one member of a pair or triplet of DNA binding dyes is fluoescent whereas the others act as counterstain, is reviewed. Appropriately chosen combinations of fluorescent dyes and counterstains can be employed to enhance general chromosome banding patterns, or to induce specific regional banding patterns. Some pairs of dyes which exhibit complementary DNA binding specificity, A-T/G-C or G-C/A-T, provide enhanced definition of positive or reverse banding patterns. Dye combinations of the type A-T/A-T, that include two DNA stains with similar specificity but non-identical binding modes, produce a specific pattern of brightly fluorescnet heterochromatic regions (DA-DAPI bands). In man, the method highlights the C bands of chromosomes 1, 9, 15, 16, and the Y. Certain dye triplets of the type G-C/A-T/A-T, which include two spectroscopically separated fluorescent stains with reciprocal DNA base pair binding specificites and a non-fluorescent A-T binding counterstain, can be used to highlight selectively, in the appropriate wavelength ranges, either R bands or DA-DAPI bands.Applications of these techniques in human cytogenetics are described. The potential of the new methodology for detecting and analysing specific chromosome bands is demonstrated. The mechanisms responsible for contrast enhancement and pattern induction are reviewed and their implications for chromosome structure are discussed as they relate to the banding phenomenon and to the DNA composition of chromosomes.     

Studies on the interaction between steffimycins and DNA.

The complexes formed between steffimycins and DNA were studied using various physicochemical techniques. The binding process has been followed spectrophotometrically or fluorimetrically. The binding parameters n and K, evaluated according to McGhee and Von Hippel, show a good affinity of these antibiotics for the macromolecule. Flow dichroism measurements showed that in the complex with DNA, the antracycline moiety of the steffimycins is intercalated between two base pairs of the macromolecule. The binding experiments with various polydeoxyribonucleotides and with various DNA samples, having different base pair compositions, suggest that an alternate sequence of A-T, such as that of poly[d(A-T)] . poly[d(A-T)], represents a good receptor site for the binding of steffimycins to DNA. The lack of in vivo activity of these antibiotics is discussed.     

Electron transfer through RNA: chemical probing of dual distance dependence

Electron transfer (ET) through RNA duplexes possessing 2'-O-pyrenylmethy uridine (Upy) and 5-bromouracil (BrU) as an electron donor and accepter set was investigated. Reductive decomposition of the BrU resulted from the ET over long distances (up to ten AU base pairs) was detected in the RNA conjugates. The RNA mediated ET from the pyrene to BrU showed dual distance dependence. This is well consistent with the previous observation for ET from Upy to nitrobenzene in RNA. In contrast, little or no reductive decomposition of the BrU was observed in the DNA conjugates when the Upy and BrU were separated by more than four AT base pairs.     

Specific interactions of divalent metal ions with a DNA duplex containing the d(CA)n/(GT)n tandem repeat

Divalent metal ions are essential for maintaining functional states of the DNA molecule. Their participation in DNA structure is modulated by the base sequence and varies depending on the nature of the ion. The present investigation addresses the interaction of Ca2+ ions with a tandem repeat of two CA dinucleotides, (CA)2/(TG)2. The binding of Ca2+ to the repeat is monitored by nuclear magnetic resonance (NMR) spectroscopy using chemical shift mapping. Parallel experiments monitor binding of Mg2+ ions to the repeat as well as binding of each ion to a DNA duplex in which the (CA)2/(TG)2 repeat is eliminated. The results reveal that the direction and the magnitude of chemical shift changes induced by Ca2+ ions in the NMR spectra of the repeat are different from those induced by Mg2+ ions. The differences between the two cations are significantly diminished by the elimination of the (CA)2/(TG)2 repeat. These findings suggest a specific interaction of Ca2+ ions with the (CA)2/(TG)2 motif. The specificity of the interaction resides in the two A-T base pairs of the repeat, and it involves the major groove of the first A-T base pair and both grooves of the second A-T base pair.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.