請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8035
標題: | 闡釋Mer2與DNA結合之生化特性在減數分裂所扮演的角色 Deciphering the role of DNA binding activity of Mer2 in meiotic DNA double-strand breaks |
作者: | Hsiao-Han Ko 柯曉涵 |
指導教授: | 冀宏源(Hung-Yuan Chi) |
關鍵字: | Mer2,減數分裂,雙股脫氧核糖核酸斷裂,同源重組,交叉, Mer2,meiosis,DNA double strand breaks,homologous recombination,chiasmata, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 減數分裂中,計畫性的雙股脫氧核糖核酸斷裂 (DNA double-strand breaks) 是必要的條件,缺乏雙股脫氧核糖核酸斷裂的染色體無法形成交叉 (chiasmata),最終染色體不能正常分配至配子中,這會導致配子中染色體數目異常,造成配子死亡。在出芽酵母菌 (budding yeast) 中,至少有十個基因參與雙股脫氧核糖核酸斷裂的形成,而Mer2 (meiotic recombination protein 2) 是其中之一。酵母菌遺傳研究發現,剔除Mer2的酵母菌其表現型為無法產生雙股脫氧核糖核酸斷裂,使減數分裂完的孢子無法存活。在Panizza (2011) 等人的文獻研究指出,在減數分裂時,Mer2會在染色體上聚集,並召集Mei4與Rec114形成複合體,最後促成雙股脫氧核糖核酸斷裂。作者更進一步推測Mer2可能具有結合雙股脫氧核糖核酸能力,但體內 (in vivo) 實驗無法證明Mer2是否能直接結合雙股脫氧核糖核酸。因此我們的實驗首先想要證實,Mer2是否具有雙股脫氧核糖核酸結合能力;其次Mer2結合雙股脫氧核糖核酸的能力,是否在減數分裂中造成雙股脫氧核糖核酸斷裂扮演重要角色。我的初步實驗結果發現:(1) 利用純化出的Mer2蛋白運用膠體電泳位移分析 (DNA mobility shift assay),證實Mer2的確能直接結合雙股脫氧核糖核酸。(2) 發現Mer2至少有兩個區域能結合雙股脫氧核糖核酸。我們進一步利用序列分析這兩段區域的鹼性胺基酸,並藉由Mer2鹼性胺基酸突變蛋白,希望在體外 (in vitro) 能找出Mer2結合雙股脫氧核糖核酸的主要鹼性胺基酸。另一方面,在體內實驗我們可以將雙股脫氧核糖核酸結合突變的Mer2基因送回缺乏Mer2的酵母菌中,觀察是否能回復孢子存活率,來證實Mer2結合雙股脫氧核糖核酸的能力,對於減數分裂產生雙股脫氧核糖核酸斷裂的重要性。 Programmed 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). Dysegulation of DSBs formation can lead to the aneuploid of inviable gamete caused by abnormal chromosome disjunction. In the budding yeast, Saccharomyces cerevisiae, there are at least ten genes mediated the formation of DSBs including Mer2. The deletion of mer2 in yeast exhibits no DSB formation and poor meiotic spore. Recent study by Panizza et al. (2011) further infers that Mer2 protein accumulates on chromosome and recruits it’s interacting partners ; Mei4 and Rec114, to promote the formation of DSBs. However, it still remains largely unknown whether Mer2 itself possesses physical interaction with double-strand DNA and whether the DNA binding activity of Mer2 is prerequisite for generating DSBs. We aim to address this question by employing highly purified recombinant Mer2 protein for in vitro DNA binding analysis. Our results indicate that Mer2 is a DNA binding protein and possesses at least two DNA binding motifs. We further narrowed down the clusters of positive charge amino acids within Mer2 that contributes to its DNA binding ability, and currently in the progression of identifying those key residues. Besides identifying DNA binding defective mutant variants of Mer2, we will also examine whether those Mer2 mutants can rescue the phenotype of poor spore viability in the deletion of mer2 strain. We have successfully generated mer2 deletion in SK1 strain, and as reported, mer2 null showed poor spore viability. In the future, we will delineate whether Mer2 DNA-binding defective mutant variants can rescue the mer2 null phenotype by complementation experiments. Our findings will shield light on the mechanism of DNA binding property of Mer2 in contributing to make DNA double-strand breaks during meiosis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8035 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2023-12-31 |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-102-1.pdf | 5.32 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。