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

Sangheeta Bhattacharjee, Julia M Richardson, Benu Brata Das

School of Biological Sciences

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Article number	102218
Number of pages	18
Journal	STAR Protocols
Volume	4
Issue number	2
Early online date	13 Apr 2023
DOIs	https://doi.org/10.1016/j.xpro.2023.102218
Publication status	Published - 16 Jun 2023

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10.1016/j.xpro.2023.102218Licence: Creative Commons: Attribution-NonCommercial-NoDerivatives (CC BY-NC-ND)

BrataDasEtalSP2023FluorescenceResonanceEnergyTransferBasedFinal published version, 4.41 MBLicence: Creative Commons: Attribution-NonCommercial-NoDerivatives (CC BY-NC-ND)

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

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. ",

author = "Sangheeta Bhattacharjee and Richardson, \{Julia M\} and Das, \{Benu Brata\}",

note = "Funding Information: B.B.D. team is supported by S Ramachandran National Bioscience Award for Career Development (NBACD)-2019 grant ( 102/IFD/SAN/3574/2019-20 ) and Indian Association for the Cultivation of Science (IACS) intramural funds. S.B. is the recipient of the IACS senior research fellowship, India. Publisher Copyright: {\textcopyright} 2023 The Author(s)",

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doi = "10.1016/j.xpro.2023.102218",

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T1 - Fluorescence-resonance-energy-transfer-based assay to estimate modulation of TDP1 activity through arginine methylation

AU - Bhattacharjee, Sangheeta

AU - Richardson, Julia M

AU - Das, Benu Brata

N1 - Funding Information: B.B.D. team is supported by S Ramachandran National Bioscience Award for Career Development (NBACD)-2019 grant ( 102/IFD/SAN/3574/2019-20 ) and Indian Association for the Cultivation of Science (IACS) intramural funds. S.B. is the recipient of the IACS senior research fellowship, India. Publisher Copyright: © 2023 The Author(s)

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Y1 - 2023/6/16

N2 - 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.

AB - 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.

U2 - 10.1016/j.xpro.2023.102218

DO - 10.1016/j.xpro.2023.102218

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JO - STAR Protocols

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ER -

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

Abstract

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