Tracing the fates of site-specifically introduced DNA adducts in the human genome

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 TIC gene. The TATAM system enabled targeting of the TK- allele with the I-Scel 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. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.orgflicenses/by-nc-nd/3.0/).