Effects of the DNA exit gate deletion onType IIA DNA topoisomerase catalysis

在生物體內，須藉由第二型DNA拓撲異構酶的催化解開參與轉錄及複製所產生的DNA超螺旋結構，及染色體濃縮與分離造成拓撲構形上的問題，為真核細胞核內必需蛋白質。 依據目前已知的結構研究及酵素動力學實驗結果，推論第二型DNA拓撲異構酶的作用機制為：酵素在N端有一缺口稱為entry gate，當酵素與一段雙股DNA(稱G-segment )結合並將之切斷，使經過entry gate進入酵素內的第二段雙股DNA(稱T-segment)通過切斷的缺口，並經過 exit gate的構形變化被送出酵素外，同時黏合G-segment缺口。目前證實第一個ATP水解驅使T-segment通過，第二個 ATP水解使酵素回覆至反應前的構形。 本實驗選用第二型DNA拓撲異構酶中胺基酸序列最短且與真核生物酵素具有相當同源性以及相同酵素活性的Phage T2 DNA拓撲異構酶為研究材料，此酵素是由T2-39(包含ATPase domain及Toprim domain )及T2-52(包含DNA cleavage/ reunion domain )兩種蛋白次單元所組成的tetramer。 在此基於第二型DNA拓撲異構酶經由長期演化所保留下來的exit gate，其作用機制目前仍不瞭解，在此利用SOE PCR技術將T2-52去除exit gate形成一內部去除蛋白經由大腸桿菌大量表現後，將蛋白質純化，再與T2-39蛋白形成突變複合物來測定其活性的變化。 在此藉由第二型DNA拓撲異構酶能利用ATP水解的能量來鬆解負超螺旋DNA的特性，以負超螺旋的質體及連環形式的kinetoplast DNA當作受質。實驗結果發現mutant enzyme具有解超螺旋作用(relaxation)及去連環活性(decatenation)是為第二型DNA拓撲異構酶。利用time course assay結果證實mutant enzyme其酵素作用仍保有processive 之特性。最後利用兩種不同作用機制的抑制劑m-AMSA 及vanadate 比較酵素對於藥物的抑制作用，實驗結果發現mutant enzyme與wild-type enzyme具有相同的inhibition pattern，因此認為其作用機制相同。TypeⅡA DNA topoisomerase are involved in all aspects of DNA metabolism including replication, transcription, recombination, and chromosome segregation. This group of enzymes manipulates DNA topology by generating a transient double-stranded break on a DNA duplex(referred as gate-forming or simply G-segment), passing the other duplex(termed transport or T-segment)through the break, and re-ligating the G-segment. The quaternary structure of all known typeⅡA topoisomerases exhibit two-fold symmetry, with two ATPase domains forming the DNA entry gate, two Toprim/CAP-like folds mediating the ATP-dependent cleavage of G-segment, and a primary dimerization region also functioning as exit gate for T-segment. In bacteriophage T2, topoisomerase Ⅱ is composed of two gene products:T2-39 for ATPase and Toprim domains, and T2-52 for CAP-like fold and the DNA exit gate. Although the structural feature of the exit gate appears to be conserved for all type ⅡA enzymes, the mechanistic roles of this observation has not yet been addressed; important molecular events such as ATP binding and hydrolysis, DNA trapping, cleavage and religation all occur in other spatially distant regions. To answer this question, we constructed an exit gate deletion mutant T2-52 using SOE PCR. The mutant protein was successfully expressed and purified to apparent homogeneity. Two assays were performed as the index for typeⅡtopoisomerase activity:supercoiled DNA relaxation and kinetoplast DNA decatenation. Surprisingly, the deletion mutant was active in both assays. We also used typical type Ⅱ topoisomerase inhibitors, vanadate and m-AMSA to analyze the mutant's sensitivity spectrum. Although the mutant was more sensitive to the inhibitor, the patterns were essentially the same. The implication of the exit gate in typeⅡ topoisomerase catalysis will be addressed in my thesis.目 錄 中文摘要 英文摘要 第一章 緒論 第一節 拓撲異構酶的生理功能與分類 第二節 第二型DNA拓撲異構酶之相關探討 第三節 本研究欲探討之方向 第二章 研究材料與方法 第一節 pET21b質體與T2-52DNA之純化 第二節 利用SOE PCR技術重組T2-52 第三節 DT2-52與pET21b的建構 第四節 酵素生化活性測定 第三章 研究結果 第四章 討論 圖表 附錄 參考文