Abasic site repair in APE1-deficient mouse embryonic stem cells: a matter for the MRN complex and ATM signaling.

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.