探討減數分裂專ㄧ重組酶Dmc1與DNA結合的動態

Abstract

同源重組反應為細胞中為相當重要的生化反應，主要功能為修補細胞中受損的DNA以維持基因的穩定性以及創造基因的多樣性。在細胞中，重組酶為負責催化同源重組反應的酵素，當細胞中的DNA產生雙股斷裂時，重組酶會在雙股斷裂處與DNA結合形成核蛋白絲、尋找並入侵同源的雙股DNA，再利用同源的雙股DNA為模版進行股交換反應，完成DNA修復。在真核生物體系中，存在有兩種重組酶：Rad51與Dmc1。在正常細胞中，Rad51會表現在有絲分裂細胞與減數分裂細胞，而Dmc1只表現於減數分裂細胞。近年來，已有眾多關於Rad51的研究，然對於Dmc1的探討卻相當有限。在本論文中，我們計畫使用單分子栓球技術，直接觀察出芽酵母菌的Dmc1與DNA結合的動態過程。我們比較Rad51與Dmc1與DNA結合動態機制，發現Rad51具有相對Dmc1優異的DNA結合能力，能在較短時間內，快速的與DNA結合形成核蛋白絲。我們也發現環境中的鈣離子可以刺激Dmc1的活性，使得Dmc1能夠更迅速地與DNA結合，為調控Dmc1活性的因子之一。

Homologous recombination (HR) plays an important role for repairing double-stranded DNA breaks in order to maintain genome integrity as well as create genetic diversity. Recombinases catalyze the HR process by first assembling on the DNA to form nucleofilaments, active components essential for HR, invading homologous duplex DNA for strand exchange and using the homologous DNA as a template to achieve DNA repair. There exist two recombinases in eukaryotic cells: Rad51, functioning in both mitotic and meiotic cells, and Dmc1, only found in meiotic cells. In spite of the extensive study of Rad51, biochemical properties of Dmc1 remain uncharacterized. In this study, we used the tethered particle motion (TPM) experiments to directly visualize the recombinase assembly on individual DNA molecules at the single molecule level. We compared the DNA assembly dynamics of Saccharomyces cerevisiae Dmc1 and Rad51 recombinases, and we found that the nucleation time of ScRad51 is far faster than that of ScDmc1. We also showed that calcium ions can stimulate ScDmc1 nucleation. Understanding the nucleofilament assembly process allows us to define how the recombinases assemble on the DNA kinetically and what the difference is between Dmc1 and Rad51.