Numerous DNA damaging events occur on a daily basis from the environments we live in, the lifestyles we lead, and through our normal metabolic processes. Damaging events that directly modify DNA bases, or cause adducts, have been linked to mutagenesis, carcinogenesis, and aging, and there is a substantial need to characterize the presence of these lesions in order to understand their role in these processes. Detection, characterization, and quantification of DNA adduct levels in cells, tissues, or individuals may provide insight into environmental exposure and allow the development of mutation signatures or biomarkers that can be correlated with disease. However, the promised outcomes of DNA adductomics has lagged behind due to technical constraints that make it difficult to monitor large numbers of adducts, utilize a variety of biological samples, and multiplex DNA adduct formation with epigenetic or genetic information. To achieve these goals, we are proposing a unique DNA adduct detection methodology, term Repair Assisted Damage Detection (R.A.D.D.) that will allow large scale detection of DNA adducts in a variety of biological samples. Our assay harnesses the highly evolved DNA repair machinery to detect adducts, then exploits DNA end labeling techniques to label or tag the sites of damage. We have established the ability of the technique to examine individual DNA adducts on isolated DNA (Zirkin, JACS 136: 7771-6, 2014), and we have now expanded its application to the detection of DNA adducts in single-cells. The unique application of fluorescent microscopy for imaging combed DNA or imaging a damaged nucleus provides novel spatial information about the formation of DNA adducts within the genome and within the nuclear compartment. It also unmasks cell to cell variability inherent in bulk techniques. To complement the single-cell data, we will also apply R.A.D.D to whole genome sequencing in order to create genome-wide maps of DNA damage hotspots. The proposed work will further extend the R.A.D.D. assay into fixed tissues, allowing DNA adduct detection across a variety of sample types, even those with limited quantities (Specific Aim 1). It will also integrate the detection of DNA adducts with precise mapping of the genome sequence where they occur and identify epigenetic marks that either occur naturally or develop as a result of the DNA adduct formation (Specific Aim 1 and 2). This novel integration of adductomics, epigenetics, and genomics will allow high resolution maps of DNA adduct sites to be developed after exposures, creating a richer information landscape of DNA damage distribution.
DNA adducts are distinct and accurate measures of individual?s exposures and their ability to detect and repair DNA damage that can cause mutations or disease. Our methodology will allow detection of DNA adducts across cells and tissues, map DNA adducts within the genome, and determine if these adducts colocalize with epigenetic DNA modifications. Together these results will inform how DNA adducts influence the initiation, development, and progress of disease.
|Gassman, Natalie R; Holton, Nathaniel W (2018) Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies. Curr Opin Biotechnol 55:30-35|
|Holton, Nathaniel W; Ebenstein, Yuval; Gassman, Natalie R (2018) Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD). DNA Repair (Amst) 66-67:42-49|