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In vitro Methylation Assay to Study Protein Arginine Methylation
Published on: October 5, 2014
Fluorescence-resonance-energy-transfer-based assay to estimate modulation of TDP1 activity through arginine
Sangheeta Bhattacharjee1, Julia M Richardson2, Benu Brata Das1
1Laboratory of Molecular Biology, School of Biological Sciences, Indian Association for the Cultivation of Science, 2A & B, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India.
Abstract:
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.

