Abstract 382: Genome-wide location analysis of DNA adducts in vivo

Abstract

Abstract DNA adducts are the biochemical consequences of exposure to UV light, carcinogens or DNA-reactive drugs. They play a central role in malignant transformation and the selective killing of cancer cells by chemotherapeutic agents. The functional consequences of DNA adducts is likely to depend on their exact location in the genome with respect to histones and sites where histones and DNA are modified. However, tools that permit precise genome-wide mapping of adduct locations have poor resolution or are too specific to the type of DNA-adduct. Here we present the development of a molecular assay (Ad-Seq) capable of identifying the genomic location of damaged DNA bases in whole cells. Using naked DNA treated with cisplatin (cDDP) or ultra-violet (UV) light as the damaging agents, the strategy relies on differential exonuclease digestion to enrich genome-wide libraries for fragments containing DNA adducts, followed by high-throughput sequencing. Consistent with the expected adduct chemistry, the 5’ end of the reads were enriched in TT, TC or CT di-nucleotides in the UV treated DNA (>4x) and in AG or GG in the cDDP treated DNA (1.5x), suggesting a positive predictive value of 76% and 44% and UV (TT) and cDDP (GG) adduct detection, respectively. Applied in vivo to IMR90 cells, Ad-Seq revealed that the fraction of AG/GG loci was higher in DNA from the cDDP-treated cells at all coverage depths, reaching 1.5x enrichment for the highest depth quartile. This indicates that the method is selective and that even low covered loci show enrichment of AG/GG sequences. The enrichment in purines was limited to 3 nucleotides downstream of the read start site, confirming the high resolution of the exonuclease digestion. A total of 11 x 103 AG/GG loci were identified in replicate in cDDP-treated samples and were absent from untreated controls. With about 60 x103 expected adducts per genome, the current assay therefore captured up to 18% of cDDP adducts. The most covered adduct sites were significantly enriched in the quiescent chromatin domains (depleted of histone modifications, p=6.7x10-31), and depleted in the transcription start sites (p=4.9x10-12), as defined by the ENCODE consortium. This may be reflective of the differences in DNA accessibility or the mechanisms of repair in these distinct regions of the genome. The analysis was performed using the PipeDuct computational package, which was developed to align the sequencing reads and provide general statistics and quality control information of Ad-Seq data. Ad-Seq is therefore a novel, unbiased method to map DNA-adducts and study in vivo the genome-wide specificity of their formation and repair. Citation Format: Brian Woo, Christopher Fang, Paolo Abada, Stephen B. Howell, Olivier Harismendy. Genome-wide location analysis of DNA adducts in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 382. doi:10.1158/1538-7445.AM2017-382