TY - JOUR
T1 - Tracing the fates of site-specifically introduced DNA adducts in the human genome
AU - Yasui, Manabu
AU - Kanemaru, Yuki
AU - Kamoshita, Nagisa
AU - Suzuki, Tetsuya
AU - Arakawa, Toshiya
AU - Honma, Masamitsu
N1 - Funding Information:
We acknowledge several helpful discussions with Dr. Takehiko Nohmi and Dr. Katsuyoshi Horibata (National Institute of Health Sciences). This work was supported by the Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology and for Health Science Foundation ( H24-food-general-011 ) from the Ministry of Health, Welfare and Labor in Japan.
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - 8-Oxoguanine (8-oxoG)
KW - DNA adducts
KW - Gene targeting
KW - Mutagenesis
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U2 - 10.1016/j.dnarep.2014.01.003
DO - 10.1016/j.dnarep.2014.01.003
M3 - Article
C2 - 24559511
AN - SCOPUS:84893721705
VL - 15
SP - 11
EP - 20
JO - DNA Repair
JF - DNA Repair
SN - 1568-7864
IS - 1
ER -