純化與闡釋裂殖酵母菌Rec12蛋白複合體的生化功能

Purification and functional characterization of S. pombe Rec12 protein complex

許家嘉(Chia-Chia Hsu)

指導教授 ： 冀宏源

國立臺灣大學/生命科學院/生化科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11380

摘要

減數分裂中，計畫性的雙股去氧核糖核酸斷裂 (DNA double-strand breaks)對於染色體正確的分離及分配到配子中是不可或缺的，而此步驟會進一步透過同源重組反應(homologous recombination)讓雙方同源的染色體彼此連結，以確保之後染色體能正確的分離及及分配到配子。缺乏雙股去氧核糖核酸斷裂的作用將會使染色體不能正常分配至配子，這會導致配子中染色體數目異常，造成配子死亡。在裂殖酵母菌 (fission yeast) 的遺傳研究中認為Rec12是個剪切計畫性雙股斷裂的重要蛋白質之一，並且發現剔除Rec12的酵母菌其表現型為無法產生雙股去氧核糖核酸斷裂，並使減數分裂後的孢子存活率大幅下降，然而由於純化Rec12有極大的困難，因此關於此蛋白質的生化特性仍有許多的未知。在我們的研究中首先想要建立一個可表現和純化重組Rec12的系統，在得到Rec12重組蛋白之後並探討其生化特性，其中包含Rec12是否具有雙股去氧核糖核酸結合能力。在這裡，我們已經成功地純化出重組Rec12蛋白複合物，並確定Rec12會和其推定的相互作用蛋白質Rec14有直接的交互作用；且重組Rec12-Rec14蛋白複合物具有雙股去氧核醣核酸的結合能力，在實驗中也證實鎂離子和三磷酸腺苷(ATP)對Rec12的去氧核醣核酸親和力並無顯著影響。然而，重組Rec12-Rec14蛋白複合體的去氧核醣核酸之裂解活性仍須做進一步的研究。

關鍵字

Rec12 ； Rec14 ； 雙股去氧核糖核酸斷裂 ； 同源重組 ； 染色體交叉

並列摘要

Programmatic double-strand breaks (DSBs) are essential for a proper chromosome segregation during meiosis, because DSBs initiate physical connection between two homologous chromosomes through homologous recombination (HR). Failure of DSBs formation can lead to the aneuploid of gamete caused by abnormal chromosome segregation. Genetic studies in Schizosaccharomyces pombe have demonstrated that the evolutionary conserved enzyme, Rec12, catalyzes the formation of DSBs. The deletion of rec12 in S. pombe exhibits poor DSB formation and decreased spore viability. However, it still remains largely unknown regarding the biochemical activity of Rec12 due to the difficulty of obtaining the recombinant protein. Thus, we aim to establish an expression and purification system for obtaining Rec12 recombinant protein and then examine its biochemical properties including DNA binding and cleavage. Here, we have successfully purified recombinant Rec12 protein complex with its putative interaction partner Rec14. We also demonstrated that Rec12 physically interacts with Rec14. Rec12-Rec14 complex possesses the DNA binding ability with the preference for dsDNA. We also documented that magnesium ions and ATP have no significant influence on the DNA binding affinity of Rec12. However, the DNA cleavage activity of purified Rec12-Rec14 complex still remains uncertain from our current study.

並列關鍵字

Rec12 ； Rec14 ； double-strand break (DSB) ； meiotic recombination ； crossover

參考文獻

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