Fluorescence-resonance-energy-transfer-based assay to estimate modulation of TDP1 activity through arginine methylation

Summary: Tyrosyl DNA phosphodiesterase (TDP1) is a DNA repair enzyme that hydrolyzes the phosphotyrosyl linkage between 3′-DNA-protein crosslinks such as stalled topoisomerase 1 cleavage complexes (Top1cc). Here, we present a fluorescence-resonance-energy-transfer-(FRET) based assay to estimate modulation of TDP1 activity through arginine methylation. We describe steps for TDP1 expression and purification and estimating TDP1 activity using fluorescence-quenched probes mimicking Top1cc. We then detail data analysis of real-time TDP1 activity and screening of TDP1-selective inhibitors.For complete details on the use and execution of this protocol, please refer to Bhattacharjee et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics