以螢光相關分光法和螢光共振能量轉移法對第二類拓撲異構酶與DNA之作用的動態分析

Abstract

拓撲異構酶(Topoisomerase)在生物體中扮演著相當重要的角色，其功能在於調控DNA的超螺旋結構，使DNA能順利被複製或轉錄成RNA，並根據其作用之DNA結構進一步分成第一型及第二型，而本文將討論第二型拓撲異構酶(Tyep II Topoisomerase, 或簡稱TopoII)。依據過去幾十年生化學家們的研究，TopoII的酵素機制為:將一條雙股螺旋DNA切斷(cleavage)，並使另一條雙股螺旋DNA通過，最後再將缺口合上(religation)。透過這樣反覆的動作，可以改變DNA拓撲結構，進而將超螺旋結構打開。然而，過去傳統的方法已無法提供更多詳細的資訊，因此近年來部份研究團隊應用光學技術來研究Top II的動力學機制，其中也包含了單分子技術。 在本研究裡，我們將介紹三種共聚焦光學顯微鏡的應用，分別為螢光相關分光法(fluorescence cross-correlation spectroscopy, FCCS)、螢光能量共振轉移法(Forster resonance energy transfer, FRET)、膠體系統單分子螢光分析。在本實驗系統中，我們設計一小段標有一對FRET螢光分子的DNA，用以觀察Top II的反應機制及動力學作用。FCCS為一單分子光學技術，藉由量測螢光分子通過偵測點時的訊號波動程度，可以決定分子的擴散速率，並進一步研究Top II和DNA的結合反應(binding reaction)。FRET則是一種能量在兩螢光分子間轉移的現象，因其轉移效率和距離相關，故能用在偵測DNA在結合(binding)或被切割(cleavage)時的結構變化；膠體系統則可用來對單一分子的螢光訊號進行分析，以得到反應的速率常數。整體來說，我們的研究提供一種更方便且快速的方法，來研究Topo II酵素反應的各個步驟，以期對Topo II有更一步的了解。

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.