Abstract 1966: In vivo detection of nucleotide lesions preceding mutation fixation

Abstract

Abstract Background and Purpose: Unrepaired DNA damage likely originates a large fraction of human cancer. However, a direct correlation between DNA damage and specific mutations is lacking. To identify in vivo nucleotide lesions that can act as the precursors of neoplastic mutations, we investigated the origin of specific tumor signature mutations previously reported on codon 122 of the p53 tumor suppressor gene (Trp53) in UVB radiation induced mouse skin tumors. Methods: The dorsal skin of wild-type (WT) and repair defective (Xpc−/− Trp53+/−) mice was shaved and exposed to a single dose of 4.8 kJ/m2 UVB radiation. Skin from unirradiated and irradiated animals was excised surgically at specific time points (0, 24 and 48 h after irradiation) and used for DNA extraction. A novel primer-anchored PCR based damage-detection methodology developed in our laboratory was used to identify and map DNA damage on the transcribed (TS) and non-transcribed strand (NTS) of the Trp53 gene. Results: Twenty-four and 48 h after UVB-radiation, significant levels of unrepaired nucleotide lesions persisted in genomic DNA of Xpc−/− Trp53+/− compared with WT mice. We observed that most of the damage blocked the primer-extension, yielding early stops. Additionally, most of the damage affecting Trp53 codon 122 in Xpc−/− Trp53+/− mice occurred in the NTS and strictly localized to 2 nucleotides. Importantly, a small percentage of the damage observed at these nucleotides allowed DNA polymerase bypass with misincorporation. Significantly, these lesions were detected precisely in the two nucleotides of Trp53 codon 122 that were found mutated in 64% of all tumors arising in this genotype. These results support the model that the tumor mutations at this site arise from error-prone bypass of long-lasting, unrepaired DNA lesions. Conclusions: We demonstrate a significant correlation between the persistence of precisely localized nucleotide lesions and the later establishment of tumor mutations at those specific nucleotides. Using a novel primer-anchored DNA damage methodology, we identified and mapped for the first time in vivo polymerase by-passable nucleotide lesions. Given the mutagenic potential of these lesions, the wide application of this novel technique for the identification of those lesions and the quantification of in vivo DNA damage might have major implications for early detection of cancer precursor lesions and evaluation of prevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1966.