Báo cáo Y học: Kinetic studies of human tyrosyl-DNA phosphodiesterase, an enzyme in the topoisomerase I DNA repair pathway

Kinetic Studies Of Human Tyrosyl-DNA Phosphodiesterase, An Enzyme In The Topoisomerase I DNA Repair Pathway

Authors: Ting-Jen Cheng, Peter G. Rey, Thomas Poon, and Chen-Chen Kan

Field: Biochemistry/Molecular Biology

Document Content: This document details the kinetic investigation of human tyrosyl-DNA phosphodiesterase (TDP), an enzyme crucial for DNA repair, particularly within the context of the topoisomerase I (Topo I) pathway. The research focuses on understanding the enzymatic mechanism of TDP and developing a high-throughput screening method for potential inhibitors. The study involves the expression and purification of recombinant human TDP variants and the development of a sensitive chromogenic assay using p-nitrophenyl-thymidine-3′-phosphate as a substrate. Kinetic parameters such as Km and Vmax were determined for human TDP, and the influence of cofactors like manganese ions was evaluated. The findings contribute to the understanding of TDP’s role in cellular processes and its potential as a therapeutic target, especially in the context of cancer treatments involving Topo I inhibitors.

Detailed Table of Contents:

• Introduction
• Experimental Procedures
• Materials
• Cloning of wild-type and mutant human TDP cDNA
• Generation of expression constructs to produce wild-type and mutant human TDP
• Production and refolding of recombinant human TDP from E. coli
• Cloning, expression, and purification of recombinant yeast TDP as the control
• Protein identification by mass spectrum analysis
• Synthesis of the chromogenic substrate
• Development and optimization of chromogenic assay for TDP
• Determination of Km, Vmax, and kcat of TDP activity
• Results
• Production of recombinant human TDP variants
• Discussion
• Acknowledgements
• References