Mapping DNA glycosylase binding across lesion sequence contexts reveals extended sequence and structural recognition logic.

DNA repair of mutagenic lesions is imperfect, allowing mutations to accumulate unevenly across the genome. In base excision repair, glycosylases must locate rare damaged bases embedded in diverse sequence contexts, yet how these contexts shape recognition and mutational outcomes remains unresolved. Here, we introduce a high-throughput approach that quantifies glycosylase binding across thousands of lesion-containing sequences. Focusing on the cytosine deamination pathway, we map the recognition landscapes of human UDG, TDG, and MBD4. Binding depends strongly on sequence context, extending several bases beyond the lesion and including non-additive interactions between neighboring positions. Structural analyses and molecular dynamics simulations implicate DNA-shape features, including minor groove width, as determinants of recognition. Nearest-neighbor preferences resemble deamination-related cancer mutational signatures, whereas broader-context preferences track variation in cytosine-thymine balance across matched human genomic contexts. Together, these findings establish a versatile and generalizable platform for decoding glycosylase recognition and linking repair specificity to mutational patterns.