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

Sangheeta Bhattacharjee; Julia M Richardson; Benu Brata Das

doi:10.1016/j.xpro.2023.102218

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

STAR Protoc. 2023 Apr 13;4(2):102218. doi: 10.1016/j.xpro.2023.102218. Online ahead of print.

Authors

Sangheeta Bhattacharjee¹, Julia M Richardson², Benu Brata Das³

Affiliations

¹ Laboratory 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.
² Institute of Quantitative Biology, Biochemistry, and Biotechnology, School of Biological Sciences, University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
³ Laboratory 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. Electronic address: pcbbd@iacs.res.in.

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.

Keywords: Cancer; Cell Biology; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Protein Expression and Purification.