title: Tracing the fates of site-specifically introduced DNA adducts in the human genome.
abstract: We developed a system for tracing DNA adducts in targeted mutagenesis (TATAM) and investigated the prevalence and types of consequent mutations. Targeted mutagenesis methods site-specifically replace endogenous DNA bases with bases carrying synthetic adducts using targeting vectors. The TATAM system was enabled by introduction of site-specific DNA double strand breaks (DSB), which strongly enhanced targeting efficiency through homologous recombination (HR), and a new polymerase chain reaction-based technique, which gives high yields of the target vectors carrying DNA adducts. Human lymphoblastoid TSCER122 cells are compound heterozygous for the thymidine kinase gene (TK-/-), and have a homing endonuclease I-SceI site in intron 4 of the TK gene. The TATAM system enabled targeting of the TK- allele with the I-SceI site using a synthetic TK+ allele containing an 8-oxo-7,8-dihydroguanine (8-oxoG) adduct, a typical product of oxidative DNA damage. The targeted clones (TK+/-) were then isolated by drug selection. Site-specific HR for DSB induced by I-SceI improved targeted integration of the synthetic allele by five orders of magnitude (from 10(-7) to 10(-2)). Subsequent analyses of approximately 800 target clones revealed that 8-oxoG was restored to G in 86% clones, probably reflecting base excision repair or translesion synthesis without mutation. Lesions of the remaining clones (14%) were associated with mutations. The mutation spectrum corresponded closely with that of oxidative DNA damage inducers reported, in which G:C to T:A transversions (5.9%) were predominant. Over-expression of MutY homologs in cells, which prevents G:C to T:A transversions by removing 8-oxoG:A mispairing, significantly decreased the frequency of mutations to 2.6%, indicating that the 8-oxoG adducts introduced by the TATAM system are processed in the same manner as those generated by oxidative DNA damage.
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Electronic address: m-yasui@nihs.go.jp.
authors.forename: Manabu
authors.initials: M
authors.lastname: Yasui
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kanemaru
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Nagisa
authors.initials: N
authors.lastname: Kamoshita
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu-cho, Hokkaido 061-0293, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Arakawa
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Electronic address: honma@nihs.go.jp.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Honma
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D002460:Cell Line; D018736:DNA Adducts / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004252:DNA Mutational Analysis; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D059645:Mutation Rate; D015183:Restriction Mapping
chemical_list: D018736:DNA Adducts; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine
keywords: 8-Oxoguanine (8-oxoG); DNA adducts; Gene targeting; Mutagenesis