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基于荧光共振能量转移的检测方法，用于评估精氨酸甲基化对TDP1活性的调节作用。

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

作者信息

Bhattacharjee Sangheeta, Richardson Julia M, Das Benu Brata

机构信息

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.

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.

出版信息

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

DOI:10.1016/j.xpro.2023.102218
PMID:37058403
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10139991/
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)..

摘要

酪氨酰-DNA磷酸二酯酶（TDP1）是一种DNA修复酶，可水解3'-DNA-蛋白质交联（如停滞的拓扑异构酶1切割复合物（Top1cc））之间的磷酸酪氨酸连接。在此，我们提出一种基于荧光共振能量转移（FRET）的测定方法，以评估精氨酸甲基化对TDP1活性的调节作用。我们描述了TDP1表达、纯化的步骤，以及使用模拟Top1cc的荧光淬灭探针评估TDP1活性的步骤。然后，我们详细介绍了TDP1实时活性的数据分析以及TDP1选择性抑制剂的筛选。有关本方案使用和执行的完整详细信息，请参阅Bhattacharjee等人（2022年）的文章。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/87bfd4c8e582/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/565debb79017/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/e16c1648955f/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/1751acfcf87a/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/8433c1ebf19c/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/0f7b1e777b9d/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/8b271cf74e9b/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/87bfd4c8e582/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/565debb79017/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/e16c1648955f/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/1751acfcf87a/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/8433c1ebf19c/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/0f7b1e777b9d/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/8b271cf74e9b/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/807e/10139991/87bfd4c8e582/gr6.jpg

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