Biochemical characterization of MIF nuclease with a FRET-based quantitative 3′ nuclease assay

Macrophage Migration Inhibitory Factor (MIF) is a PARP1-associated nuclease involved in DNA replication, emerging as a promising therapeutic target due to its elevated expression in various tumor types. We developed a FRET-based quantitative nuclease assay to specifically measure MIF's cleavage of 3′ overhangs. This assay identified Mg 2+ as an essential cofactor, with optimal activity at 10 mM, and revealed a dual modulatory role of glucose. This platform enables robust enzymatic characterization and high-throughput screening of inhibitors, offering a critical tool for advancing anticancer drug development.