Archaeal HhH-GPD proteins expand the functional repertoire of DNA glycosylases in base excision repair.

Genomic DNA continuously sustains oxidative, alkylative and deaminative lesions that compromise genome stability in all three domains of life. Base excision repair (BER), the major conserved pathway eliminating small non-bulky base damage, is initiated by substrate-specific DNA glycosylases. The helix-hairpin-helix-Gly/Pro/Asp (HhH-GPD) superfamily comprises ancient, functionally diverse glycosylases ubiquitous across Bacteria, Eukarya and Archaea. Prior cross-kingdom reviews outline general HhH-GPD features, but no dedicated work systematically catalogues and phylogenetically analyzes archaeal HhH-GPD homologs. This comprehensive review solely focuses on archaeal HhH-GPD proteins, covering four canonical subfamilies (EndoIII, OGG2/AGOG, AlkA, and MIG) and two divergent atypical enzymes: an euryarchaeal HhH-GPD-fold uracil DNA glycosylase from and a crenarchaeal alkylation-specific HhH-GPD with a non-canonical active site. Integration of AlphaFold3 predictions and solved crystal/NMR structures dissects conserved core folds and lineage-specific structural adaptations. Further analysis of Asgard archaeal HhH-GPD homologs traces eukaryotic BER evolutionary origins. Distinct from earlier cross-domain reviews, this review focuses specifically on archaeal HhH-GPD proteins, emphasizing unique structural features, expanded non-canonical paralogs and catalytic versatility. Outstanding research gaps and tiered future avenues are outlined, alongside unique mechanistic and evolutionary insights exclusive to archaeal HhH-GPD systems.