DNA modifications drive aging, neurodegeneration, carcinogenesis and chemotherapy drug action. Accurate mapping of diverse DNA modifications with single-nucleotide precision in complex genomes remains challenging. We upgraded click-code-seq, a click-chemistry-aided DNA-modification mapping strategy, to enable its first application for sequencing oxidation and depurination in the human genome. We developed a companion fluorescence assay, click-fluoro-quant, to rapidly quantify common DNA modifications and novel adaptors to minimize false positives and assess modification frequency. We uncovered that endogenous DNA oxidation in a human cell line mirrors cancer mutational signatures linked to oxidative stress. The chemotherapy drug irofulven preferentially induces depurination in ApA dimers and promoters. Notably, oxidized guanines and apurinic sites, both irofulven induced and endogenous, are depleted in gene transcribed strands, with the strand bias increasing with gene expression. This work substantially advances click-code-seq for deciphering the impacts of key modifications in human DNA on cellular physiology and toxicological responses.

Ultraviolet (UV) radiation from the sun causes adverse skin changes such as premature aging. UV-induced mitochondrial DNA (mtDNA) alterations, including deletions, contribute to photoaging and cellular dysfunction. While the most frequent mtDNA rearrangement is the common deletion (CD), characterized by the loss of nearly one-third of the genome (4977 bp), detailed knowledge of mechanisms governing UV-mediated initiation of the CD and mitigation strategies are lacking. Here, we investigated how increasing UV exposure increases CD levels in human skin fibroblasts via cellular reactive oxygen species (ROS) formation and mtDNA oxidation and demonstrated that antioxidant preconditioning of cells prevents UVA-induced CD accumulation. Conversely, UVB exposure induced cyclobutane pyrimidine dimers (CPDs) without affecting ROS, suggesting an ROS-independent pathway. Using a 3D full-thickness human skin model, we confirmed UVA-dependent CD formation in both the epidermis and dermis. RNA-Seq analysis of UVA-exposed fibroblasts revealed upregulation of mitochondrial DNA replication genes and downregulation of mtDNA repair genes. These findings provide insight into how UVA and UVB differ in detrimental effects on mtDNA, with UVA impacting mtDNA maintenance and transcription via a ROS-dependent mechanism, and provide a physiologically relevant platform to evaluate potential interventions.

Ultraviolet (UV) radiation from the sun causes adverse skin changes such as premature aging. UVA radiation is the primary factor for photoaging due to its deep penetration into the dermis, and UV-induced mitochondrial DNA (mtDNA) alterations, including deletions, contribute to photoaging and cellular dysfunction. The most frequent mtDNA rearrangement is the common deletion (CD), characterized by the loss of nearly one-third of the genome, 4,977 base pairs. UV radiation exposure leads to the formation of the CD, however, a distinct characterization of UV-induced CD and the underlying molecular mechanisms driving its initiation remains unexplored. In this study, we showed that increasing doses of UV radiation led to an increase in the CD in human skin fibroblasts. We found that UVA induce the formation of the CD by increasing the cellular reactive oxygen species (ROS) and oxidized bases content in the mtDNA. Preconditioning cells with antioxidants prevented the accumulation of the UVA-induced CD, suggesting that this mutational mechanism is ROS-dependent. In stark contrast, UVB did not alter cellular ROS levels but increased the formation of cyclobutane pyrimidine dimers (CPD), leading to CD generation though a ROS-independent mechanism. We corroborated our findings by using a 3D human full-thickness skin equivalent model, where we detected UVA-dependent CD formation in both the epidermal and dermal layers of the skin. By analyzing bulk RNA from UVA-exposed human skin fibroblasts by RNA-Seq, we found that UVA led to the upregulation of genes encoding mitochondrial DNA replication proteins and to the downregulation of genes involved genes encoding mtDNA repair factors. Taken together, our findings provide insight into how UVA and UVB differ in their detrimental effects on mtDNA, with UVA impacting mtDNA maintenance and transcription via a ROS-dependent mechanism. Our findings also established the mtDNA CD as a novel potential biomarker for monitoring UVA-induced oxidative stress and photoaging in skin cells in vitro and in vivo.

The dual role of base excision repair (BER) in resolving DNA base lesions and enabling active DNA demethylation positions BER proteins as central regulators of both genetic and epigenetic integrity. While BER and active demethylation converge at the generation of AP sites, catalyzed by DNA glycosylases such as thymine DNA glycosylase (TDG), the relative contribution of each pathway to AP site formation and the cellular fate of these lesions remain unresolved. Using APE1-deficient mouse embryonic stem cells (mESCs), with inducible TDG depletion, we reveal that TDG activity accounts for a substantial fraction of AP sites in mESCs, establishing a direct link between active DNA demethylation and APE1-mediated BER. We demonstrate that APE1 is essential for mESC differentiation, underscoring its role in processing AP sites generated during epigenetic reprogramming. Through a genome-wide CRISPR/Cas9 screen designed to map the broader functional interactome of APE1, we identified unexpected genetic dependencies that extend beyond DNA repair. We also found that DNA double-strand break repair factors act outside their canonical pathways, cooperating with nucleotide excision repair and translesion synthesis to compensate for the loss of APE1. These findings highlight novel compensatory DNA end-processing mechanisms that safeguard genomic integrity in the absence of APE1, revealing a high level of plasticity of DNA repair in mESCs.