Dataset Information

Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate.

ABSTRACT: Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of aqueous solutions of DNA, during which *OH is one of the major ROS, can lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32P-postlabeling studies suggested that the intrastrand cross-link lesions may arise from Fenton reaction, which also induces the formation of *OH; the structures of the proposed intrastrand cross-link lesions, however, have not been determined. Here, we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H2O2/ascorbate could lead to the formation of an intrastrand cross-link lesion, i.e., G wedge T, where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G wedge T. In addition, the yield of the intrastrand cross-link was approximately 3 orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-formyl-2'-deoxyuridine. The induction of intrastrand cross-link lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

SUBMITTER: Hong H

PROVIDER: S-EPMC2519820 | biostudies-other | 2006 May

REPOSITORIES: biostudies-other

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Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate.

Hong Haizheng H Cao Huachuan H Wang Yuesong Y Wang Yinsheng Y

Chemical research in toxicology 20060501 5

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of aqueous solutions of DNA, during which *OH is one of the major ROS, can lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32P-postlabeling studies suggested that the intrastrand cross-link lesions may ...[more]

PMID: 16696563

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Project description:Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.

Project description:Nucleotide excision repair (NER) plays an important role in maintaining the integrity of DNA by removing various types of bulky or distorting DNA adducts in both prokaryotic and eukaryotic cells. In Escherichia coli, the excision repair proteins UvrA, UvrB, and UvrC recognize and incise the bulky DNA damages induced by UV light and chemical carcinogens. In this process, when a putative lesion in DNA is identified initially by UvrA, a subsequent strand opening is carried out by UvrB that not only ensures that the distortion is indeed due to a damaged nucleotide but also recognizes the chemical structure of the modified nucleotides with varying efficiencies. UvrB also recruits UvrC that catalyzes both the 3'- and the 5'-incisions. Herein, we examined the interaction of UvrABC with a DNA substrate containing a single G[8,5-Me]T cross-link and compared it with T[6,4]T (the 6-4 pyrimidine-pyrimidone photoproduct) and the C8 guanine adduct of N-acetyl-2-aminofluorene (AAF). The intrastrand vicinal cross-link G[8,5-Me]T containing a covalent bond between the C8 position of guanine and the 5-methyl carbon of the 3'-thymine is formed by X-radiation, while T[6,4]T is a vicinal cross-link induced by the UV light. We also selected the AAF adduct for comparison because it represents a highly distorting monoadduct containing a covalent linkage at the C8 position of guanine. The dissociation constants (K(d)) for UvrA protein binding to DNA substrates containing the G[8,5-Me]T, T[6,4]T, and AAF adducts, as determined by gel mobility shift assays, were 3.1 +/- 1.3, 2.8 +/- 0.9, and 8.2 +/- 1.9, respectively. Although UvrA had a considerably higher affinity for G[8,5-Me]T than for the AAF adduct, the G[8,5-Me]T intrastrand cross-link was incised by UvrABC much less efficiently than the T[6,4]T intrastrand cross-link and the AAF adduct. Similar incision results also were obtained with the DNA substrates containing the adducts in a six-nucleotide bubble, indicating that the inefficient incision of G[8,5-Me]T cross-link by UvrABC was probably due to the lack of efficient recognition of the adduct by UvrB at the second step of DNA damage recognition in the E. coli NER. Indeed, as compared to T[6,4]T and AAF substrates, which clearly showed UvrB-DNA complex formation, very little UvrB complex was detectable with the G[8,5-Me]T substrate. Our result suggests that G[8,5-Me]T intrastrand cross-link is more resistant to excision repair in comparison with the T[6,4]T and AAF adducts and thus will likely persist longer in E. coli cells.

Dataset Information

Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate.

Publications

Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate.

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