Structural Connectivity Between the Zinc Linchpin Motif, the [4Fe-4S] cluster, and the active site orchestrates DNA repair in MUTYH

The DNA glycosylase MUTYH protects genomic integrity by excising adenine mispaired with 8-oxoguanine (OG), which initiates base excision repair (BER). The [4Fe-4S] cluster DNA binding domain and Zn linchpin motif of MUTYH are hotspots for inherited cancer associated variants (CAVs) highlighting their critical functions in DNA repair. Here, we present three full-length human MUTYH crystal structures bound to DNA across three catalytic states, representing early and late transition states and product complexes. These structures reveal a previously unrecognized interplay between the Zn site and the Fe–S cluster mediated by His85 and a conserved Arg247/Arg307 bridge spanning ∼15 Å. Disruption of this network impairs metal loading, reduces the active enzyme fraction, weakens lesion DNA binding, and diminishes OG:A repair in cells. Moreover, the Zn site adopts distinct coordination modes across the reaction coordinate, with a water molecule replacing a cysteine ligand in transition state analog complexes and cysteine coordination restored in a product-mimicked state. Functional analyses show that this ligand switching is dispensable for core glycosylase chemistry in vitro but disproportionately affects repair in cells, suggesting an additional role of the Zn linchpin in cellular OG:A repair beyond intrinsic glycosylase activity, likely involving interactions with BER partners. Together, these findings illustrate how the Zn linchpin enhances Fe–S cluster domain DNA substrate engagement to enable effective DNA repair and provides a rationale for how these functions are compromised by MUTYH CAVs.