Singlet oxygen-induced DNA damage: product analysis, studies of biological consequences and characterization of mutations.
Lutgerink Jtvan den Akker EDaniëlle PachenSmeets EjJohannes van DijkAubry JmHans JoenjeLafleur MvJ. Retèl
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The DNA lesions induced by free 1O2 and the biological and mutagenic consequences of 1O2-induced DNA damage have been studied. Using anion exchange HPLC, reverse-phase HPLC with electrochemical detection and 32P-postlabelling methods, we have shown that 1O2 reacts with 2'-deoxyguanine 3'-monophosphate (dGp) but not with any other dNp. Reaction with dGp yields a large number of products; one minor product was identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp), and a second tentatively as a formamidopyridine derivative of dGp. 8-Oxo-dGp was also found after reaction of 1O2 with single-stranded (ss) DNA, double-stranded (ds) DNA or an oligonucleotide (16-mer) having one G. With the oligonucleotide we found a second unidentified reaction product. With ss DNA, 8-oxo-dG was a much more prominent product than in the reaction of 1O2 with free dGp and the yield was about eight-fold higher than with ds DNA. This agrees with our finding that ss M13 DNA is at least 100-fold more sensitive than ds M13 DNA to biological inactivation by 1O2. The inactivation of ss M13 DNA must be largely due to 1O2-induced lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG, most of the mutations induced by 1O2 in ss or ds M13mp10 DNA occurred at a G or G/C basepair, respectively. A preference for G(C) to T(A) transversions was observed for which 8-oxo-dG might have been responsible. In ss DNA, a significant number of mutations are characterized by the fact that a G is deleted.Keywords:
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In the present work, we study the reaction of singlet oxygen (1O2) with isolated DNA. Emphasis is placed on the identification and quantitative measurement of the DNA modifications that are produced by the reaction of 1O2 with DNA. For this purpose, calf-thymus DNA was incubated with the endoperoxide of N,N′-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide, a chemical generator of 1O2. Thereafter, DNA was digested, and the resulting oxidized nucleosides were measured by means of a recently optimized high-performance-liquid-chromatography tandem-mass-spectrometry assay. It was found that, among the different DNA lesions observed, 7,8-dihydro-8-oxo-2′-deoxyguanosine is the major 1O2-mediated DNA-damage product. Interestingly, cyclobutane pyrimidine dimers, oxidized pyrimidine bases, 7,8-dihydro-8-oxo-2′-deoxyadenosine, and 2,6-diamino-5-formamido-4-hydroxypyrimidine are not formed, at least not in detectable amounts, following treatment of DNA with the 1O2 generator. The reported results strongly suggest that the decomposition of the endoperoxide provides a pure source of 1O2, and that reaction of 1O2 with isolated DNA induces the specific formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine.
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Photobiology
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Deoxyadenosine
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We previously demonstrated that ultraviolet (UV) light (254 nm) induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA via a singlet oxygen mechanism. In the present paper, we provide novel findings that DNA structure and base composition significantly affect the yield of 8-OHdG by UV radiation. Unlike ionizing radiation that induces 8-OHdG both in free 2'-deoxyguanosine (dG) and in DNA, UV light induced 8-OHdG formation in intact DNA and polydG·dC, but not in dG. When thermally denatured DNA was irradiated with UV light, the yield of 8-OHdG was reduced by more than 80% compared to intact DNA. Oxygenation of the denatured DNA solution did not restore the yield of UV-induced 8-OHdG. Irradiation of DNA with different AT/GC ratios showed that the yield of UV-induced 8-OHdG varied in proportion to the AT content, suggesting that AT base pairs in DNA enhance generation of the oxidizing species and subsequent oxidation of dG. The natural antioxidants genistein, estradiol, protocatechuic acid (PCA), and oleanolic acid (OA) were investigated for their inhibition of UV-induced 8-OHdG. Genistein and estradiol, that intercalate into DNA as shown by a computer modeling, significantly quenched UV-induced 8-OHdG, whereas PCA and OA did not fit into DNA and exhibited weak or no effect. These results suggest that the intercalation of genistein and estradiol into DNA may alter the DNA structural integrity, interrupt the production of oxidizing species, and subsequently reduce the formation of 8-OHdG by UV radiation.
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The DNA lesions induced by free 1O2 and the biological and mutagenic consequences of 1O2-induced DNA damage have been studied. Using anion exchange HPLC, reverse-phase HPLC with electrochemical detection and 32P-postlabelling methods, we have shown that 1O2 reacts with 2'-deoxyguanine 3'-monophosphate (dGp) but not with any other dNp. Reaction with dGp yields a large number of products; one minor product was identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp), and a second tentatively as a formamidopyridine derivative of dGp. 8-Oxo-dGp was also found after reaction of 1O2 with single-stranded (ss) DNA, double-stranded (ds) DNA or an oligonucleotide (16-mer) having one G. With the oligonucleotide we found a second unidentified reaction product. With ss DNA, 8-oxo-dG was a much more prominent product than in the reaction of 1O2 with free dGp and the yield was about eight-fold higher than with ds DNA. This agrees with our finding that ss M13 DNA is at least 100-fold more sensitive than ds M13 DNA to biological inactivation by 1O2. The inactivation of ss M13 DNA must be largely due to 1O2-induced lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG, most of the mutations induced by 1O2 in ss or ds M13mp10 DNA occurred at a G or G/C basepair, respectively. A preference for G(C) to T(A) transversions was observed for which 8-oxo-dG might have been responsible. In ss DNA, a significant number of mutations are characterized by the fact that a G is deleted.
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Porphyrins have been studied as photosensitizers in photodynamic therapy. DNA is one of the most important targets of the sensitizer. In the present study, we have examined the photosensitized DNA damage caused by dihydroxo P ( V ) tetraphenylporphyrin ( P ( V ) TPP ), a cationic water-soluble porphyrin. P ( V ) TPP photosensitized guanine-specific damage to the DNA fragment. P ( V ) TPP induced severe photodamage to single-stranded rather than to double-stranded DNA. High performance liquid chromatography measurements confirmed the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-G), an oxidized product of 2'-deoxyguanosine, and showed that the content of 8-oxo-G in single-stranded DNA is larger than that in double-stranded DNA. The effects of reactive oxygen scavengers on DNA damage suggested the involvement of singlet oxygen ( 1 O 2 ). Photosensitized 1 O 2 formation was confirmed by near-infrared emission measurements. The results showed that 1 O 2 formation mainly contributes to the mechanism of DNA photodamage by P ( V ) TPP . Absorption spectrum measurements showed the interaction between P ( V ) TPP and DNA. This interaction is expected to enhance the 1 O 2 -mediated DNA damage since the lifetime of 1 O 2 in a cell is very short. On the other hand, P ( V ) TPP induced DNA damage at the consecutive guanines in double-stranded DNA. Because the consecutive guanines act as a hole trap, this DNA-damaging pattern suggests the partial involvement of photo-induced electron transfer. The fluorescence of P ( V ) TPP was quenched by DNA, supporting the electron transfer mechanism. However, DNA damage by electron transfer was not a main mechanism possibly due to reverse electron transfer. In conclusion, P ( V ) TPP binds to DNA and induces guanine-specific, photo-oxidation mainly via 1 O 2 generation.
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7,8‐Dihydro‐8‐oxoguanine (8‐oxoguanine; 8‐oxo‐G), one of the major oxidative DNA adducts, is highly susceptible to further oxidation by radicals. We confirmed the higher reactivity of 8‐oxo‐G toward reactive oxygen (singlet oxygen and hydroxyl radical) or nitrogen (peroxynitrite) species as compared to unmodified base. In this study, we raised the question about the effect of this high reactivity toward radicals on intramolecular and intermolecular DNA damage. We found that the amount of intact nucleoside in oligodeoxynucleotide containing 8‐oxo‐G decreased more by various radicals at higher levels of 8‐oxo‐G incorporation, and that the oligodeoxynucleotide damage and plasmid cleavage by hydroxyl radical were inhibited in the presence of 7,8‐dihydro‐8‐oxo‐2′‐deoxyguanosine (8‐oxo‐dG). We conclude that 8‐oxo‐G within DNA induces intramolecular DNA base damage, but that free 8‐oxo‐G protects intermolecular DNA from oxidative stress. These results suggest that 8‐oxo‐G within DNA must be rapidly released to protect DNA from overall oxidative damage.
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Abstract 8‐Oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxo‐dG) is a mutagenic DNA lesion which is prone to further oxidation. One‐electron oxidants like Ir IV oxidize 8‐oxo‐dG to give secondary lesions such as the spiroiminodihydantoin. This lesion blocks DNA polymerases and induces G → T as well as G → C transversions. Here, we report the synthesis of a carbocyclic analog of the 8‐oxo‐dG lesion and the carbocyclic version of the spiroiminodihydantoin lesion. Both lesion analogs were obtained within single‐stranded DNA and as their corresponding nucleosides. Characterization of the lesion analogs was achieved by HPLC, ESI‐MS and ESI‐MS/MS, as well as enzymatic digestion of the oligonucleotides. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)
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