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.
Highlights
Genomic DNA is constantly exposed to both exogenous and endogenous genotoxic agents
The protein expression was evaluated by Western blot analysis with anti-xeroderma pigmentosum group A (XPA) monoclonal antibody (Fig 2B), showing that XPA protein was expressed in TSCER122 but not in XPA KO cells
OGG1, APE1, and polymerase β (Pol β) were detected in both TSCER122 and XPA KO cells, which indicates that the disruption of XPA did not significantly alter the expression level of base excision repair (BER) components
Summary
Genomic DNA is constantly exposed to both exogenous and endogenous genotoxic agents. Among them, ionizing radiation (IR) induces various DNA adducts in the genome because of PLOS ONE | DOI:10.1371/journal.pone.0142218 November 11, 2015. It is more challenging to repair 8-oxoG in clustered DNA damage sites via BER. Different types of damage (a thymine glycol, an AP site, a single-strand break, or a mismatched base-pair) adjacent to 8-oxoG strongly inhibits 8-oxoG excision by OGG1 [8,9,10]. Several studies have examined the mutagenic events of clustered oxidative damage to episomal DNA in mammalian cells [25, 26], these repair mechanisms in the human genome are still not well understood. On the basis of these studies, we posed the following question: what role does NER play in the suppression of mutagenesis induced by a single and/or clustered 8-oxoG formed in the genome?. Our findings indicate that NER is a possible repair mechanism of clustered oxidative DNA adducts in TS of the human genome
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