title: Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.
abstract: Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sassa
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Nagisa
authors.initials: N
authors.lastname: Kamoshita
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kanemaru
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Honma
authors.affiliation: Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Yasui
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair; D055786:Gene Knockout Techniques; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D016296:Mutagenesis; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C453560:7,8-dihydro-8-oxoguanine; D018736:DNA Adducts; D012333:RNA, Messenger; D051760:Xeroderma Pigmentosum Group A Protein; D006147:Guanine