Abstract
Background/Objectives: Uracil-DNA glycosylase (UNG) initiates base excision repair by removing uracil from DNA. Although Family 1 UNGs are well characterized in model organisms, the biochemical and functional properties of Staphylococcus aureus UNG (SaUNG) is not well characterized. This study aimed to characterize SaUNG. Methods: Recombinant wild-type and mutant SaUNG proteins were examined using biochemical and kinetic assays, site-directed mutagenesis, genetic complementation in Escherichia coli, and in vitro reconstitution of S. aureus base excision repair. Results: SaUNG exhibited maximal activity at pH 8.0–8.5, 37–45 °C, and 100–150 mM KCl and lacked AP-lyase activity. Catalytic efficiencies (kcat/Km) were 1.39 × 109, 3.79 × 108, and 2.93 × 108 M−1·min−1 for ssU, U:G, and U:A, respectively, establishing the preference ssU > U:G > U:A. H180A retained residual activity, whereas simultaneous substitution of Asp59 and His180 abolished detectable activity. Complete inhibition confirmed conserved biochemical properties of Family 1 UNGs. SaUNG complemented the mutation avoidance defect of an E. coli (ung− mug−) mutant and initiated uracil repair in a reconstituted S. aureus base excision repair system. Conclusions: SaUNG is a canonical Family 1 UNG with experimentally characterized biochemical properties, catalytic determinants, and biological function in uracil repair.