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.     

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.     

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.     

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.     

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 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.