Photosensitized formation of 8-hydroxydeoxyguanosine in cellular DNA by riboflavin.

Photosensitized formation of 8-hydroxydeoxyguanosine (oh8dG) in DNA by riboflavin has recently been shown in vitro. The present study describes the formation of oh8dG in cellular DNA by photo-irradiation of cultured mammalian cells in the presence of riboflavin. Formation of oh8dG was dependent on the concentration of riboflavin as well as the duration of photoirradiation. These results suggest that photosensitized formation of oh8dG in DNA by riboflavin may be involved in photocarcinogenesis.     

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.     

Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA.

Membrane lipid peroxidation processes yield products that may react with DNA to cause oxidative modifications. We have investigated this possibility and have found that calf thymus DNA exposed to autooxidized lipids causes the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 8-OH-dG formation in DNA was measured using high-pressure liquid chromatography with electrochemical detection. Methyl linolenate oxidized for different lengths of time was exposed to DNA. The amount of 8-OH-dG formed in DNA was proportional to the amount of lipid peroxidation as measured by the thiobarbituric reactive substances present. The formation of 8-OH-dG in DNA by autooxidized methyl linolenate was dependent on the presence of the transition metal ions Cu or Fe and was inhibited by various scavengers, including superoxide dismutase and catalase. This implicates the involvement of oxygen free radicals in the process. Liposomes formed from phosphatidylcholine (82%) and methyl arachidonate (18%) were peroxidized for different lengths of time and then exposed to DNA. 8-OH-dG was formed in DNA by exposure to Cu(II) and peroxidized liposomes. Under these conditions, Fe(III) was slightly less effective than Cu(II) in mediating 8-OH-dG formation. These observations clearly show that 8-OH-dG formation in DNA may result from processes that may occur during intracellular lipid peroxidation.     

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.     

Age-associated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diaphragm

This is the first report that age-associated accumulation of 8-hydroxydeoxyguanosine (8-OH-dG) does occur in human mitochondrial DNA (mtDNA) in muscle of diaphragm. We extracted mtDNA from human diaphragm muscles from differing age groups, and determined the amount of 8-OH-dG by ultramicro-high performance liquid chromatography/mass-spectrometry system. With the same specimen, multiple deletions of mtDNA were detected by electrophoresis after amplification by the polymerase chain reaction method. In subjects below age 55, the level of 8-OH-dG in mtDNA was below 0.02% of the total deoxyguanosine (dG), whereas, in subjects over age 65, the level of 8-OH-dG increased with age at a rate of ca. 0.25% per 10 years, reaching 0.51% at age 85. Moreover, a concomitant increase in multiple deletions was detected with the increase in age. These results suggest that, in younger diaphragms, replication of mtDNA dilutes out 8-OH-dG being not detectable. In the elderly subjects aged over 65, the replication rate might be slowed down leading to the accumulation of 8-OH-dG in mtDNA, which would accelerate the age-associated multiple deletions of mtDNA observed among the subjects.     

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

NMR Studies of a DNA Containing 8-Methoxydeoxyguanosine

Abstract

¹H NMR experiments have been undertaken to elucidate the structural effects of methoxy substitution at the C8 of a deoxyguanosine residue in a self-complementary dodecadeoxyribonucleotide, d(C-G-C-mo⁸G-A-A-T-T-C-G-C-G), duplex, which has an 8-methoxy-2′-deoxyguanosine (mo⁸dG) residue at the 4th position. NMR data indicate that the mo⁸dG residue takes an anti glycosidic conformation in a mo⁸dG(anti):dC(anti) base-pair structure in a B-form duplex. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(C-G-C-G-A-A-T-T-C-G-C-G) duplex.     

NMR solution structure of a DNA dodecamer containing single G.T mismatches.

The three-dimensional solution structure of the self-complementary DNA dodecamer (CGT_GACGT_TACG above GCAT_TGCAG_TGC] which contains the thermodynamically destabilizing [TG_A above AT_T] motif was determined using two-dimensional NMR spectroscopy and simulated annealing protocols. Relaxation matrix analysis methods were used to yield accurate NOE derived distance restraints. Scalar coupling constants for the sugar protons were determined by quantitative simulations of DQF-COSY cross-peaks and used to determine sugar pucker populations. Twenty refined structures starting from random geometries converged to an average pairwise root mean square deviation of 0.49 A. Back calculated NOEs give Rc and Rx factors of 0.38 and 0.088, respectively. The final structure shows that each of the single G@T mismatches form a wobble pair with two hydrogen bonds where the guanine projects into the minor groove and the thymine projects into the major groove. The incorporation of the destabilizing [TG_A above AT_T] motif has little effect on the backbone torsion angles and helical parameters compared to standard B-form duplexes, which may explain why G.T mismatches are among the most commonly observed in DNA. The structure shows that perturbations caused by a G.T mismatch extend only to its neighboring Watson-Crick base pair, thus providing a structural basis for the applicability of the nearest-neighbor model to the thermodynamics of internal G.T mismatches.     

1H NMR studies of lac-operator DNA fragments.

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.     

Formation of 8-hydroxydeoxyguanosine in DNA treated with fecapentaene- 12 and -14

Fecapentaene-12 and -14, direct-acting mutagens in human feces, were found to hydroxylate the C-8 position of guanine residues in DNA in vitro. Fecapentaene-12 or -14 was incubated with 0.5 mg of calf thymus DNA in 1 ml of reaction mixture at pH 7.4 for 2 h at 37°C in the dark, and then 8-hydroxydeoxyguanosine (8-OH-dG) was analyzed. In these conditions 8-OH-dG was formed dose-dependently at levels of 1.1–4.6 residues/10⁴ dG with concentrations of 0.5–3.0 mM of fecapentaene-12. Similar results were obtained with fecapentaene-14. The amount of 8-OH-dG in untreated DNA was 0.2–0.3 residue/10⁴ dG.     

Formation of 8-hydroxydeoxyguanosine in DNA treated with fecapentaene-12 and -14

Fecapentaene-12 and -14, direct-acting mutagens in human feces, were found to hydroxylate the C-8 position of guanine residues in DNA in vitro. Fecapentaene-12 or -14 was incubated with 0.5 mg of calf thymus DNA in 1 ml of reaction mixture at pH 7.4 for 2 h at 37 degrees C in the dark, and then 8-hydroxydeoxyguanosine (8-OH-dG) was analyzed. In these conditions 8-OH-dG was formed dose-dependently at levels of 1.1-4.6 residues/10(4) dG with concentrations of 0.5-3.0 mM of fecapentaene-12. Similar results were obtained with fecapentaene-14. The amount of 8-OH-dG in untreated DNA was 0.2-0.3 residue/10(4) dG.     

Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics.

Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5' end of the tetra-loop has been solved by NMR spectroscopy. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T(12)and U(15)nucleotide stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and U(15)while T(13)partially stacks upon T(14). Very weak stacking interaction is observed between T(13)and T(12). All the individual nucleotide bases adopt ' anti ' conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T(13)and T(14). The stereochemistry of U(15)mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5' end, as reported previously.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

NMR solution structure of a DNA dodecamer containing a transplatin interstrand GN7-CN3 cross-link.

The DNA duplex d(CTCTCG*AGTCTC).d(GAGAC-TC*GAGAG) containing a single trans- diammine-dichloroplatinum(II) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR. All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5'/H5") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined. The duplex is distorted over two base pairs on each side of the interstrand cross-link and exhibits a slight bending of its axis ( approximately 20 degrees ) towards the minor groove. The platinated guanine G* adopts a syn conformation. The rotation results in a Hoogsteen-type pairing between the complementary G(6)* and C(19)* residues which is mediated by the platinum moiety and is stabilized by a hydrogen bond between O6(G(6)*) and N4H(C(19)*). The rise between the cross-linked residues and the adjacent residues is increased owing to the interaction between these adjacent residues and the ammine groups of the platinum moiety. These results are discussed in relation to the slow rate of closure of the monofunctional adducts into interstrand cross-links.     

Multinuclear NMR studies of DNA hairpins. 2. Sequence-dependent structural variations

The solution conformation of three related DNA hairpins, each with five bases in the loop, is investigated by proton and phosphorus 2D NMR methods. The sequences of the three oligomers are d(CGCGTTGTTCGCG), d(CGCGTTTGTCGCG), and d(CTGCTCTTGTTGAGCAG). One pair of hairpins shares the same stem sequence but differs in the loop, and the appearance of an unusual phosphate torsion in the stem is found to depend on the sequence in the loop of the hairpin. The second pair of hairpins shares the same loop region but differs in the stem sequence in that the base pair which closes the loop is a C-G or G-C pair. The pattern of NOEs reveals that the stacking arrangement in the loop region depends on the base pair that closes the stem. These results suggest that hairpin loop conformation and dynamics are sensitive to small changes in the loop and adjacent stem sequences. These findings are discussed in relation to sequence-dependent thermodynamic changes that have been observed in RNA hairpins.     

Duplex-hairpin transitions in DNA: NMR studies on CGCGTATACGCG.

Two dimensional NMR methods have been used to assign proton resonances in the high salt (greater than or equal to 100mM Na+), low temperature duplex form of the self-complementary DNA dodecamer d(CGCGTATACGCG). At low salt (less than or equal to 10mM Na+) and higher temperature marked changes in the two-dimensional spectrum, and in the one-dimensional spectrum reported by others, indicate that the molecule converts to an alternate conformation. Using saturation transfer methods, many of the resonances of this new conformation have been assigned, and the kinetics of the interconversion of the two forms has been studied. The linewidth, correlation time, and concentration dependence of the formation of this alternate conformation support the idea that it is a unimolecular hairpin. Observation of chemical shifts and NOEs in the hairpin conformation allow some preliminary structural characterization. Examination of the energetics of the interconversion suggests that the exchange between forms does not proceed through a single stranded intermediate, but rather through another pathway, probably involving a cruciform structure.     

Detection and quantitation of 8-hydroxydeoxyguanosine 5'-monophosphate in X-irradiated calf-thymus DNA by fluorescence postlabeling

8-Hydroxydeoxyguanosine 5'-monophosphate (8-OH dGmp) was synthesized from deoxyguanosine 5'-monophosphate (dGmp) by ascorbic acid in the presence of hydrogen peroxide and labeled with dansyl chloride through a phosphoramidate linkage with ethylenediamine (EDA). A DNA model 8-OHd(TACG), isolated intact by high pressure liquid chromatography (HPLC) from x-irradiated d(TACG) and characterized by nmr, was digested enzymatically to 5'-mononucleotides. The modified nucleotide was enriched by HPLC and dansylated. Analysis of the dansylated product by HPLC, using a fluorescent detector, detected a peak with retention time corresponding to that of the dansyl labeled authentic marker. The same overall procedure was used to detect 8-OHdGmp from x-irradiated calf-thymus DNA. The content of 8-OHdGmp in the irradiated DNA increased linearly with increasing levels of x-irradiation in the dose range of 6-60 Gy.     

NMR and CD studies on an oligonucleotide containing N4-methylcytosine.

The hexamer d(CGm4CGCG) exists predominantly as a right handed B form helix at 20 degrees C in 150 mM NaCl, as shown by 2D NOE spectra. Under these conditions a minor species is also observed which corresponds to the single strand in slow exchange on a proton NMR time scale with the double strand. This exchange is unusually slow and separate resonances for the two species are seen up to 65 degrees C. At 50 degrees C the lifetime of the single strand species is 0.85 s. Under high salt conditions the hexamer is partly converted into the Z form, but the complete transition is only observed at 5M NaCl at -6 degrees C.     

Oxidative DNA damage (8-hydroxydeoxyguanosine) and body iron status: a study on 2507 healthy people

To clarify the relationship of oxidative stress and body iron status, we detected urinary 8-hydroxydeoxyguanosine (8-OHdG) as a biomarker of oxidative DNA damage, and measured serum ferritin and total iron-binding capacity (TIBC), both reflecting body iron store, on 2507 healthy people aged between 22 and 89 years (males, 1253; females, 1254). The urinary 8-OHdG excretion of males showed almost no change with age, but the excretion of premenopausal females was lower than that of males, whereas postmenopausal females excreted significantly more than males. The values of serum ferritin showed no remarkable change with age in males, but increased gradually in postmenopausal females without iron loss due to bleeding, although the males' values remained higher than those of females at all ages (p<.05). On the other hand, the values of TIBC remained within the narrow limits in males, regardless of age, whereas those of females always stayed at a higher level than the males (p<.05). Conclusively, urinary 8-OHdG correlated with serum ferritin positively and with TIBC inversely, which suggested that body iron status would control the generation of 8-OHdG in vivo. After all, the increase of urinary 8-OHdG excretion in postmenopausal females may be caused by the decrease of body iron loss.     

Molecular modeling studies of a parallel stranded quadruplexes containing a 8-bromoadenosine

Truncated sequences of human telomeric DNA can readily assemble to form parallel stranded quadruplexes containing A- and G-tetrads. The formation of an A-tetrad is highly context-dependent and the relationship between the formation of an A-tetrad and the glycosidic torsion angle of the adenosine residues implicated has not been completely clarified so far. In order to give a further insight in this issue we synthesized the modified oligomers d(ABrGGGT) and d(TABrGGGT), two different truncations of the human telomeric sequence containing a 8-bromoadenosine residue, named ABr. NMR data show that both the modified oligomers are able to perfectly fold into highly symmetric quadruplexes with all strands parallel to each other. Molecular modeling studies were performed on both [d(ABrGGGT)]4 and [d(TABrGGGT)]4, indicating that a bulky substituent, such as a bromine atom at the C8 position of adenines, can force the glycosidic bond to adopt a syn conformation, stabilizing the resulting quadruplexes.