Abstract
Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts. Here, we generated a mutant Af1521 macrodomain fused to green fluorescent protein (GFP) to generate a high-affinity ADP-ribosyl binding reagent. The resulting tool – which we call MacroGreen – is easily produced by expression in Escherichia coli, and can detect both mono-and poly-ADP-ribosylation of diverse proteins in vitro. Staining with MacroGreen allows detection of ADP-ribosylation at sites of DNA damage by fluorescence microscopy. MacroGreen can also be used to quantify modification of target proteins in overlay assays, and to screen for PARP inhibitors in high-throughput format with excellent assay statistics. We expect that this broadly applicable tool will facilitate ADP-ribosylation related discoveries, including by laboratories that do not specialize in this field.