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DNA damage measurements within tissue samples with Repair Assisted Damage Detection (RADD).

ABSTRACT: Exposures to genotoxic carcinogens and reactive species result in strand breaks and a spectrum of covalent modifications to DNA that can induce mutations and contribute to the initiation and progression of cancer. Measurements of DNA damage within tissue or tumor samples can serve as a biomarker for exposures or assess changes in DNA repair capacity relevant in cancer development and treatment. Numerous methods to characterize DNA damage exist. However, these methods are primarily applicable to isolated DNA or cultured cells, often require a substantial amount of material, and may be limited to the detection and quantification of only a handful of DNA adducts. Here, we used the Repair Assisted Damage Detection (RADD) assay to detect and excise DNA adducts using a cocktail of DNA repair enzymes, then the damage site within the genome are tagged for detection using a modified nucleotide. We previously demonstrated the RADD assay can detect lesions within isolated DNA and fixed cells, and now RADD can be used to detect DNA adducts and DNA strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue samples. We verified the ability of the RADD assay to detect DNA damage in tissue by exogenously inducing DNA damage with X-rays and restriction enzymes. We also showed that RADD can be multiplexed with antibodies to detect cell cycle markers or other proteins of interest. Finally, we showed that RADD can detect DNA damage within clinically relevant ovarian tumor samples. RADD is a flexible and easy-to-use assay that allows relative damage levels to be determined within FFPE samples and allows the heterogeneity of DNA adducts and strand breaks within clinically relevant samples to be measured.

SUBMITTER: Lee KJ

PROVIDER: S-EPMC8118132 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:DNA is damaged by various endogenous and exogenous sources, leading to a diverse group of reactive intermediates that yield a complex mixture of products. The initially formed products are often metastable and can react to yield lesions that are more biologically deleterious. Mechanistic studies are frequently carried out on free DNA as the substrate. The observations do not necessarily reflect the reaction environment inside human cells where genomic DNA is condensed as chromatin in the nucleus. Chromatin is made up of monomeric structural units called nucleosomes, which are comprised of DNA wrapped around an octameric core of histone proteins (two copies each of histones H2A, H2B, H3, and H4).This account presents a summary of our work in the past decade on the mechanistic studies of DNA damage and repair in reconstituted nucleosome core particles (NCPs). A series of metastable lesions and reactive intermediates, such as abasic sites (AP), N7-methyl-2'-deoxyguanosine (MdG), and 2'-deoxyadenosin-N6-yl radical (dA•), have been independently generated in a site-specific manner in bottom-up-synthesized NCPs. Detailed mechanistic studies on these NCPs revealed that histones actively participate in DNA damage and repair processes in diverse ways. For instance, nucleophilic residues in the flexible histone N-terminal tails, such as Lys and N-terminal α-amine, react with electrophilic DNA damage and reactive intermediates. In some cases, transient intermediates are produced, leading to the promotion or suppression of damage and repair processes. In other examples, reactions with histones yield reversible or stable DNA-protein cross-links (DPCs). Histones also utilize acidic and basic residues, such as histidine and aspartic acid, to catalyze DNA strand cleavage through general acid/base catalysis. Alternatively, a Tyr in histone plays a vital role in nucleosomal DNA damage and repair via radical transfer. Finally, the reactivity discovered during the mechanistic studies has facilitated the development of new reagents and methods with applications in biotechnology.This research has enriched our knowledge of the roles of histone proteins in DNA damage and repair and their contributions to epigenetics and may have significant biological implications. The residues in histone N-terminal tails that react with DNA lesions also play pivotal roles in regulating the structure and function of chromatin, indicating that there may be cross-talk between DNA damage and repair in eukaryotic cells and epigenetic regulation. Also, in view of the biased amino acid composition of histones, these results provide hints about how the proteins have evolved to minimize their deleterious effects but maximize beneficial ones for maintaining genome integrity. Finally, previously unreported DPCs and histone post-translational modifications have been discovered through this research. The effects of these newly identified lesions on the structure and function of chromatin and their fates inside cells remain to be elucidated.

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DNA damage measurements within tissue samples with Repair Assisted Damage Detection (RADD).

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