Kinetic Analysis of DNA Binding of Topoisomerase II by FCCS and FRET
Date Issued
2014
Date
2014
Author(s)
Lee, Chun-Ying
Abstract
Type II topoisomerases are essential enzymes that are involved in chromosome segregation and structural modifications, and in regulating intracellular DNA supercoiling. In the past decades, the enzymatic function of topoisomerase II has been widely studied by biochemists. It acts by transiently breaking one DNA double strand (cleavage), allowing the second double strand to pass through the opened DNA gate, followed by rejoining the broken ends (religation) to close the gate. However, part of the mechanisms still remains unclear. Recently, optical techniques have been applied to investigate the kinetics of topoisomerase, especially single molecule methods. In this study, we describe three applications of confocal spectroscopy: fluorescence cross-correlation spectroscopy (FCCS), Forster resonance energy transfer (FRET) in solution, and single molecule FRET analysis in gel-based system. In our system, we use a unique oligonucleotide substrate with a pair of fluorophore straddling the topoisomerase II cleavage site to monitor the kinetic behavior of this enzyme. FCCS is a biophysical technique that measures fluorescence fluctuations in a confined focal volume to determine diffusion coefficient, and thus, the binding kinetics of DNA and topoisomerase II can be addressed. FRET monitors the distance between a pair of fluorophores on the DNA. The energy transfer efficiency depends on the DNA conformational change induced by the enzyme binding. Both solution and gel-based FRET system can be used to investigate the dynamics of DNA gate during the catalytic cycle. To sum up, our method provides an opportunity to study the individual steps in a Top II catalytic cycle: to measure the binding by FCCS and to monitor catalytic reactions by FRET.
Subjects
拓撲異構酶
螢光相關分光法
螢光能量共振轉移法
單分子螢光分析
Type
thesis
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