Abstract C13: A novel DNA damage detection assay for prediction of individual cancer risk

Abstract

Abstract Background: Unrepaired DNA damage leads to a host of diseases including cancer. Despite the profound impact of DNA damage on human health, little progress has been made in the development of assays capable of accurately measuring induced as well as endogenous in vivo DNA damage. We developed a novel primer-anchored DNA damage detection assay (PADDA) to map and quantify a broad range of DNA lesions at the nucleotide level. Our damage detection assay relies on the principle that the presence of DNA damage on the template can change the polymerase replicative rate, leading to lesion bypass with or without misincorporation, or to lesion-dependent replicative arrest before or opposite the damaged base. Methods: PADDA's specificity, sensitivity and dynamic range were first determined on artificial templates containing engineered DNA lesions and on hydrogen peroxide damaged DNA. To determine the sensitivity of PADDA to detect different spectra of in vivo DNA lesions, we mapped and quantified endogenous and induced DNA damage in wild-type (wt) and repair defective yeast and mice cells. In vivo spontaneous DNA lesions were mapped within the CAN1 gene of S. cerevisiae wt and base excision repair (BER) defective cells and compared to the location of CAN1 published mutations. In vivo induced DNA lesions were mapped within the TRP53 gene in wt and nucleotide excision repair mutant (Xpc−/−) mice irradiated with ultraviolet B (UVB) light. The location of nucleotide lesions were compared to the published p53 mutational spectrum for identical strains. Results: PADDA screens a specific DNA region for base damage, provides detailed fingerprint analysis of base damage and repair at the singlenucleotide level, and quantifies strand-specific DNA damage on a high-throughput scale. The sensitivity of PADDA to detect and map engineered lesions within synthetic oligonucleotides was 100% for abasic and 8-oxodA lesions, 85% for 8-oxoG lesions, and 67% for thymine glycol lesions. PADDA was also able to quantify in vitro induced DNA damage over a wide range of genotoxic exposures (1 μM to 500 mM hydrogen peroxide). Furthermore, PADDA detected significantly higher levels of endogenous damage in yeast cells in stationary phase than in exponential phase, and in yeast BER defective cells than in isogenic wt cells at any phase of growth. Importantly, PADDA detected levels of endogenous DNA lesions that are consistent with published estimates. When mapping endogenous DNA damage, several clusters of DNA lesions were identified in the CAN1 gene. Fisher's exact test showed that the damaged nucleotides detected by PADDA were associated with those previously reported to be the sites of mutations in CAN1. Similarly, a correlation was found between persistent UVB induced DNA damage in the non-transcribed strand of Xpc-/- mice and a previously reported mutational hotspot. These data demonstrate that PADDA is capable of identifying pre-mutagenic DNA lesions in vivo. Conclusion: PADDA is the first assay capable of accurately mapping and quantifying nucleotide and strand specific endogenous DNA damage. PADDA's unprecedented ability to map sites of persistent in vivo endogenous and induced DNA damage before mutation fixation and to quantify strand specific damage may become a critical tool for assessing cancer risk and prevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C13.