UV-DDB as a Dynamic Regulator Linking Base Excision and Nucleotide Excision Repair via AAG Interaction

Base excision repair (BER) and nucleotide excision repair (NER) are traditionally regarded as independent pathways; however, accumulating evidence indicates that ultraviolet (UV)-damaged DNA-binding protein (UV-DDB), a core NER factor, stimulates BER DNA glycosylases, including alkyladenine DNA glycosylase (AAG). Despite this functional link, the molecular basis of the UV-DDB/AAG interaction and its regulation by DNA remain unclear. This study investigated the direct interaction between AAG and UV-DDB using electrophoretic mobility shift assays (EMSA), surface plasmon resonance (SPR), biolayer interferometry (BLI) and AlphaFold3-based structural modeling under DNA-free and DNA-bound conditions. SPR analysis revealed that AAG and UV-DDB form a high-affinity complex in the absence of DNA (KD ≈ 17.5 nM), which is maintained but reduced approximately 2.6-fold upon binding to apurinic/apyrimidinic site (AP site)-containing dsDNA (KD ≈ 46.2 nM). BLI analysis independently confirmed this interaction under both DNA-free and DNA-bound conditions, with inter-platform differences consistent with previously reported BLI/SPR variability. EMSA showed UV-DDB-mediated ternary complex formation accompanied by redistribution of binary AAG/DNA species. AlphaFold3 modeling predicted that AAG associates with DDB1 in the DNA-free state, whereas under DNA-bound conditions, DDB2 recognizes the AP site while AAG repositions toward the lesion with multiple active site residues placed in close proximity. These findings support a model in which DNA binding acts as a molecular switch that reconfigures the UV-DDB/AAG interaction, potentially enabling UV-DDB to function as a recruitment platform that facilitates directional progression of AAG through the BER cycle, and providing a structural basis for coordinated integration of BER and NER.