"探討植物阿拉伯芥中，減數分裂時期之類拓樸異構酶VI (the meiosis-specific topoisomerase VIB-like protein), MTOPVIB, 的生化特性"

Abstract

在進行減數分裂時，同源重組 (homologous recombination) 對於同源染色體能否正確分配到子細胞中及遺傳訊息的彼此互換，扮演著不可或缺的角色。減數分裂重組反應的發生，主要是藉由DNA雙股斷裂來啟動。在過去的研究中，SPO11已經被充分證明是造成DNA雙股斷裂的重要酵素，更被認為是屬於第II型拓樸異構酶VI (type II topoisomerase VI, Topo VI) 中A次單元 (subunit) 的同源物。目前已知拓撲異構酶VI是由A和B兩個次單元所組成，可藉由ATP的結合與水解形成循環達到調節DNA拓樸構型之目的。然而，至今卻沒有生化實驗的證據可以證實 SPO11單獨存在即可在體外 (in vitro) 造成DNA雙股斷裂；以及是否需要類Topo VIB蛋白 (Topo VIB-like protein) 的協助，才能使SPO11具有造成DNA雙股斷裂的酵素活性。直到最近，在阿拉伯芥 (A. thaliana) 植物中找到了減數分裂時期的類Topo VIB蛋白，MTOPVIB。透過結構模擬的方式，MTOPVIB和古細菌S. shibatae Topo VIB具有高度的結構相似性；值得注意的是，MTOPVIB不但會與SPO11蛋白產生交互作用，也參與在減數分裂時期DNA雙股斷裂的形成。但是，MTOPVIB的生化特性至今仍是尚未被解開的謎，舉凡：(1) MTOPVIB是否具有結合並水解ATP的活性？(2) 在MTOPVIB存在的情況下可否活化SPO11的酵素活性？為了深入研究這些問題，我們首先成功的純化出MTOPVIB及MTOPVIB-SPO11複合物。我們的研究結果指出，MTOPVIB蛋白本身是具有結合DNA的能力，並對雙股DNA有較高的親和力；然而，MTOPVIB不具和ATP結合的能力，並且無法活化SPO11造成DNA雙股斷裂的活性。有趣的是，我們的研究結果猜測類Topo VI複合物 (topoisomerase VI-like complex) 與雙股DNA的結合能力可能主要是由MTOPVIB次單元所提供。綜觀上述，我們的研究成果成功的闡釋A. thaliana MTOPVIB和MTOPVIB-SPO11複合物的生化特性，並提供SPO11與典型type II topoisomerase VI在反應機制上可能的差異。

Meiotic recombination is essential for proper chromosome segregation and for shuffling genetic information during meiosis. SPO11, the type II topoisomerase VI A-like protein, has been well documented to generate DNA double-strand breaks (DSBs) for the initiation of meiotic recombination. Topoisomerase VI consists of A and B subunits to regulate DNA topology through the cycle of ATP binding and hydrolysis. However, no biochemical evidence shows whether SPO11 alone is sufficient to make DSBs in vitro and whether TopoVIB-like protein is a prerequisite for activation of SPO11-mediated DNA cleavage. Recently, Arabidopsis thaliana topoisomerase VI B-like protein, MTOPVIB (meiosis-specific topoisomerase VIB-like), is identified as a structural homolog of archaeon S. shibatae Topo VIB subunit. Importantly, MTOPVIB interacts with SPO11 and is essential for the DSBs formation during meiosis. It remains enigmatic whether MTOPVIB is akin to its Topo VIB ortholog harboring ATPase activity and activating SPO11 activity. Here, we successfully purify the recombinant MTOPVIB protein and MTOPVIB-SPO11 complex. Our results reveal that MTOPVIB is a bona fide DNA-binding protein with a preference for duplex DNA, but lacks the ability of binding nucleotide cofactor ATP and the activation of SPO11-mediated DNA cleavage in vitro. Interestingly, the results suggest that MTOPVIB is the major DNA-binding subunit of topoisomerase VI-like complex. In conclusion, our findings provide insights into the biochemical characteristics of A. thaliana MTOPVIB and MTOPOVIB-SPO11 complex. Importantly, the mechanistic divergence between SPO11 and classical type II topoisomerase is also elucidated in this study.