Biochemical and structural studies reveal the substrate specificity and catalytic mechanism of MYG1 as a two-metal ion-dependent 3′→5′ exonuclease

• MYG1 is a Mn2+- or Mg2+-dependent 3′→5′ exonuclease with broad substrate specificity, showing highest activity on single-stranded RNA and a preference for U in RNA and dT in DNA. • Structural and biochemical analyses reveal that MYG1 functions as a dimer, with the active site formed by the catalytic domain of one monomer and the substrate-binding domain of the other, and employs a two-metal ion catalytic mechanism. • The study identifies key residues involved in metal ion and substrate binding, providing insights into the molecular basis of MYG1's substrate specificity and catalytic activity. • These findings lay a foundation for understanding the biological roles of MYG1 in cellular processes and its potential implications in human diseases, offering a target for future therapeutic interventions.