title: 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/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.
authors.affiliation: Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Haizheng
authors.initials: H
authors.lastname: Hong
authors.forename: Huachuan
authors.initials: H
authors.lastname: Cao
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D003596:Cytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005720:Gamma Rays; D006147:Guanine / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D053719:Tandem Mass Spectrometry
chemical_list: D006147:Guanine; D003596:Cytosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003851:Deoxyribonucleases