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

Manabu Yasui, Yuki Kanemaru, Nagisa Kamoshita, Tetsuya Suzuki, Toshiya Arakawa, Masamitsu Honma

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)11-20
Number of pages10
JournalDNA Repair
Volume15
Issue number1
DOIs
Publication statusPublished - 2014 Mar 1
Externally publishedYes

Fingerprint

Mutagenesis
DNA Adducts
Human Genome
Genes
Clone Cells
DNA
DNA Damage
Mutation
Homologous Recombination
Alleles
Double-Stranded DNA Breaks
Thymidine Kinase
Deoxyribonuclease I
Polymerase chain reaction
Mutation Rate
DNA Repair
Introns
Repair
Polymerase Chain Reaction
8-hydroxyguanine

Keywords

  • 8-Oxoguanine (8-oxoG)
  • DNA adducts
  • Gene targeting
  • Mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tracing the fates of site-specifically introduced DNA adducts in the human genome. / Yasui, Manabu; Kanemaru, Yuki; Kamoshita, Nagisa; Suzuki, Tetsuya; Arakawa, Toshiya; Honma, Masamitsu.

In: DNA Repair, Vol. 15, No. 1, 01.03.2014, p. 11-20.

Research output: Contribution to journalArticle

Yasui, Manabu ; Kanemaru, Yuki ; Kamoshita, Nagisa ; Suzuki, Tetsuya ; Arakawa, Toshiya ; Honma, Masamitsu. / Tracing the fates of site-specifically introduced DNA adducts in the human genome. In: DNA Repair. 2014 ; Vol. 15, No. 1. pp. 11-20.
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