Mapping the sequence logic of DNA repair enzyme binding reveals mechanistic principles and evolutionary links

Mutations compromise genome stability, promote disease, and yet drive genetic diversity and evolution. DNA repair of mutagenic lesions acts to maintain genome integrity but is inherently imperfect, allowing mutations to emerge, persist and accumulate unevenly across the genome.Understanding when and where such mutations arise requires a deep understanding of the molecular factors that govern repair enzyme recognition. In base excision repair (BER), glycosylases must locate rare damaged bases that appear within diverse sequence and structural contexts across the genome, yet how these contexts modulate recognition and influence mutational outcomes remains unresolved.Here we introduce a high-throughput approach that quantifies BER-glycosylase binding across thousands of lesion-containing sequence contexts. Focusing on the cytosine-deamination pathway, we mapped the recognition landscapes of the human enzymes UDG and TDG, which act on the modified base uracil and on T:G mismatches derived from cytosine and 5-methylcytosine deamination, respectively. Our results reveal widespread sequence- and structure-dependent influences on binding, extending several bases from the lesion site and including non-additive interactions between flanking positions. Structural analyses implicate DNA-shape features such as base-step Slide and minor-groove width as determinants of recognition. Notably, sequence preferences influenced by flanks beyond the immediate neighbors reflect the cytosine–thymine (C/T) balance in the human genome, revealing a striking connection between long-range binding specificity and localized mutation patterns.Together, these findings and framework establish a generalizable approach for dissecting how DNA repair enzymes recognize lesions in sequence- and structure-specific contexts, providing a foundation for predictive models of how repair fidelity shapes genome evolution and human disease.