Formation and genotoxicity of a guanine–cytosine intrastrand cross-link lesion in vivo

Abstract

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/γ-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to γ-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 109 nucleosides per Gy, which was ∼300 times lower than that of 5-formyl-2′-deoxyuridine (0.011 lesions per 106 nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G→T and a 1.2% G→C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo.

Efficient and accurate bypass of N 2 -(1-carboxyethyl)-2′-deoxyguanosine by DinB DNA polymerase in vitro and in vivo

“…4A), underscoring the lack of involvement of pol II and V in bypassing the N 2 -CEdG in vivo. We also measured the bypass efficiencies by using the recently introduced LC-MS/MS method (32), and the results are in keeping with those measured by using the conventional CRAB assay (Fig. S4).…”

“…S12) (28). (31,32). To determine bypass efficiency, we also prepared a competitor genome by inserting a 19-mer unmodified ODN, d(GAAGACCAGCTAGCT-GCGG), into the linearized M13 genome.…”

“…Restriction fragments were subjected to LC-MS/MS analysis, and a gradient of 5 min of 0Ϫ20% methanol followed by 35 min of 20 -50% methanol in 400 mM hexafluoro-2-propanol buffer (pH was adjusted to 7.0 by the addition of triethylamine) was used for the separation (32,38,39). The mass spectrometer was set up for monitoring the fragmentation of the [M-2H] 2Ϫ ions of the 8-and 11-mer ODNs.…”

Mutagenic and Cytotoxic Properties of 6-Thioguanine, S-Methylthioguanine, and Guanine-S6-sulfonic Acid

“…2) (27,28). In this respect the restriction digestion mixture was analyzed by LC-MS/MS, and we moni- …”

Limited Repair of Critical DNA Damage in Cells Exposed to Low Dose Radiation

“…As for the data on the repair of IR-induced ICL, they are rather limited. This is most likely caused by methodological difficulties of their acquisition against a variety of other IR-induced DNA lesions and, in particular, because of their association with clustered lesions (Ding and Greenberg, 2007;Hong et al, 2007;Gantchev et al, 2009). It should be noted that the cells of patients with the syndrome of Fanconi anemia (FA) are characterized by genome instability, and predisposition to malignancy, and by increased sensitivity to IR due to their inability to repair ICL of DNA (Alter, 2002;Kuhnert et al, 2009).…”