Formation and genotoxicity of a guanine-cytosine intrastrand cross-link lesion in vivo - PubMed (original) (raw)

Haizheng Hong et al. Nucleic Acids Res. 2007.

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/gamma-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 gamma-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 10(9) nucleosides per Gy, which was approximately 300 times lower than that of 5-formyl-2'-deoxyuridine (0.011 lesions per 10(6) 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.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Identification of a G[8-5]C intrastrand cross-link lesion in HeLa-S3 cells. Shown are the SICs for the m/z 555→457→261 (a) and m/z 558→460→264 (b) transitions for the in vivo formed intrastrand cross-link and the isotope-labeled internal standard, respectively. Depicted in (c) is the structure for the isotope-labeled internal standard (bold italic letters designate the 15N- and 13C-labeling sites). Panel (d) reveals the dose-responsive formation of the G[8-5]C lesion.

Figure 2.

Figure 2.

The REAMS assay for the monitoring of the in vivo cytotoxicity and mutagenicity of the G[8-5]C lesion. For the ligation scaffolds, only the portions of the sequences that are complementary to the 16-mer lesion insert are shown, while the remaining sequences are indicated by black lines. The PCR products and restriction fragments for the competitor genome are not listed. BbsI and Tsp509I cleavage sites are indicated by solid and broken arrows, respectively, and the recognition sites for these two restriction endonucleases are highlighted in bold.

Figure 3.

Figure 3.

LC-MS/MS for monitoring the restriction fragments of interest without mutation or with a G→T mutation at the original guanine portion of the lesion [i.e. d(GCGACGCC) and d(TCGACGCC)]. Shown in (a) and (b) are the SICs for the formation of indicated fragment ions of these two ODNs, and illustrated in (c) and (d) are the MS/MS of the [M–2H]2− ions of these two ODNs (m/z 1196.9 and 1184.3). Nomenclature for fragment ions follow that described by McLuckey et al. (56).

Figure 4.

Figure 4.

In vivo bypass efficiencies and mutation frequencies of DNA lesions in wild-type AB1157 E. coli cells determined by the REAMS assay. The data represent the means and standard deviations of results from three independent transformation and LC-MS/MS experiments.

Figure 5.

Figure 5.

In vivo bypass efficiencies and mutation frequencies of G[8-5]C in pol II-, pol IV- and pol V-deficient AB1157 E. coli determined by REAMS assay. The data represent the means and standard deviations of results from three independent transformation and LC-MS/MS experiments.

Similar articles

Cited by

References

    1. Finkel T., Holbrook N.J. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239–247. - PubMed
    1. Lindahl T. DNA lesions generated in vivo by reactive oxygen species, their accumulation and repair. NATO ASI Ser., Ser. A: Life Sci. 1999;302:251–257.
    1. Marnett L.J. Oxyradicals and DNA damage. Carcinogenesis. 2000;21:361–370. - PubMed
    1. Bellon S., Ravanat J.L., Gasparutto D., Cadet J. Cross-linked thymine-purine base tandem lesions: synthesis, characterization, and measurement in gamma-irradiated isolated DNA. Chem. Res. Toxicol. 2002;15:598–606. - PubMed
    1. Box H.C., Budzinski E.E., Dawidzik J.B., Gobey J.S., Freund H.G. Free radical-induced tandem base damage in DNA oligomers. Free Radic. Biol. Med. 1997;23:1021–1030. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources