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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 冀宏源(Hung-Yuan Chi) | |
dc.contributor.author | Yi-Cheng Chen | en |
dc.contributor.author | 陳奕成 | zh_TW |
dc.date.accessioned | 2021-06-15T11:14:56Z | - |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49061 | - |
dc.description.abstract | 在減數分裂中,同源染色體的配對和同源重組對於同源染色體正確的分配到子細胞中是必要的。不正常的同源染色體配對和同源重組會導致非整倍體(aneuploid)的配子(gametes)產生。在分裂酵母(Schizosaccharomyces pombe)中,線性單元(linear elements, LinEs)能幫助同源染色體的配對,並藉由調控Rec12蛋白質產生雙股斷裂(double-strand breaks, DSBs)來起始同源重組反應。喪失參與線性單元中的分子會導致無法正常的產生去氧核醣核酸(DNA)雙股斷裂和配對同源染色體。Fowler和Smith(2013)等人的研究指出線性單元分子:Rec27,Rec25和Mug20對於Rec12產生雙股斷裂的熱點(hot spots)是必須的。作者更進一步推測Rec27,Rec25和Mug20可以形成蛋白質複合體(complex)並促進Rec12的酵素活性而產生DNA雙股斷裂。因此我想要藉由純化Rec27,Rec25和Mug20蛋白並分析他們的生化特性去探討和Rec12的關係。
我們藉由大腸桿菌(E. coli)表現並純化Rec27,Rec25和Mug20重組蛋白。我們初步藉由體外親和性沉降(in vitro affinity pull down)、酵母菌雙雜合分析(yeast two-hybrid analysis)以及膠體電泳位移分析(DNA mobility shift assay)發現:(1) Rec27,Rec25和Mug20之間有直接的交互作用;(2) 只有Rec27擁有和雙股去氧核醣核酸(DNA)結合的能力。除此之外,我們也成功建立了多順反子(polycistronic)表現系統,同時表現並純化Rec27-Rec25-Mug20複合體。我們的研究可以用來進一步分析Rec27-Rec25-Mug20複合體和Rec12產生的雙股斷裂之間的關係。 | zh_TW |
dc.description.abstract | Homologous pairing and meiotic recombination are essential for proper chromosome segregation during meiosis. Failures of pairing and recombination can lead to the aneuploid of gametes. In Schizosaccharomyces pombe, linear elements (LinEs) are responsible for homologous pairing and programmed DNA double-strand breaks (DSBs) catalyzed by conserved Rec12 protein to initiate meiotic recombination. Loss of LinEs components leads to defects of pairing and DSBs formation. It has been well documented that only Rec27, Rec25, and Mug20 are enriched at DSB hotspots and required for hot spots formation. Those observations suggested that Rec27, Rec25, and Mug20 can form a complex and activate Rec12-mediated DSBs (Fowler et al., 2013). This work aims to investigate the biochemical property of Rec25, Rec27, and Mug20, and their functional relationship with Rec12-mediated DSB.
We used E. coli as host cells to express and purify Rec27, Rec25, and Mug20 recombinant proteins. We demonstrated that Rec27, Rec25, and Mug20 have a physical interaction and importantly, only Rec27 harbors a DNA binding ability. We also established the polycistronic expression system to co-express and purify Rec27-Rec25-Mug20 complex to study their biochemical characteristics. Our study provides useful information for further to decipher the functional role of Rec27-Rec25-Mug20 complex in Rec12-mediated DSBs. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:14:56Z (GMT). No. of bitstreams: 1 ntu-105-R02b46002-1.pdf: 3738836 bytes, checksum: 8e0a7e045a8d8cef10fb9b4eca928b50 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | CHAPTER 1: Introduction
1-1. The biology of meiosis……………………………………………………………………… 1 1-2. The importance of homologous pairing and meiotic recombination……………………… 3 1-3. The synaptonemal complex (SC) formation and meiotic recombination……………………… 3 1-4. The regulation between AEs and meiotic recombination……………………… 5 1-5. Conserved meiotic DSB formation in Schizosacharomyces pombe……………………… 6 1-6. Linear elements (LinEs) are responsible for homologous chromosomes pairing in Schizosacharomyces pombe, like SC in other species………………… 9 1-7. Novel LinEs proteins, Rec27, Rec25, and Mug20 can affect the formation of meiotic DSBs………………………………………………………………… 10 1-8. Motivation of my study……………………………………………………... 12 CHAPTER 2: Materials and Methods 2-1. DNA substrates……………………………………………………………… 13 2-1-1. 80nt single-strand DNA (ssDNA)……………………………………. 13 2-1-2. 80bp double-strand DNA (dsDNA)………………………………….. 13 2-2. E. coli and Yeast strains……………………………………………………... 13 2-2-1 E. coli strains…………………………………………………………. 13 2-2-2 Yeast strains…………………………………………………………... 14 2-3. Plasmids…………………………………………………………………….. 16 2-3-1 Protein expression plasmids………………………………………….. 16 2-3-1-1 Rec27 expression plasmid…………………………………… 16 2-3-1-2 Rec25 expression plasmid…………………………………… 16 2-3-1-3 Mug20 expression plasmid…………………………………... 16 2-3-1-4 Rec27, Rec25, and Mug20 complex expression plasmid……. 17 2-3-1-5 Rec27 DNA binding mutant plasmids……………………….. 17 2-3-2 Yeast two hybrid plasmids……………………………………………. 18 2-3-2-1 GAL4 DNA binding domain (BD) fusion protein plasmids… 18 2-3-2-2 GAL4 activation domain (AD) fusion protein plasmids…….. 18 2-4. Rec27, Rec25, and Mug20 Protein Expression and Purification…………… 18 2-4-1 Expression of Rec27-MBP, TrxA-Rec25, and NusA-Mug20………... 18 2-4-1-1 Rec27-MBP expression and purification…………………….. 18 2-4-1-2 TrxA-Rec25 expression and purification…………………….. 20 2-4-1-3 NusA-Mug20 expression and purification…………………... 22 2-4-1-4 MBP-Rec25 expression and purification…………………….. 23 2-4-1-5 Rec27 DNA binding mutants expression and purification…... 25 2-4-2 Polycistronic expression of Rec27-MBP, TrxA-Rec25, and NusA -Mug20……………………………………………………………….. 25 2-5. In vitro affinity pull-down assay……………………………………………. 27 2-5-1. S-protein agarose pull-down………………………………………… 27 2-5-2. Amylose resin pull down…………………………………………….. 28 2-6. DNA mobility shift assay…………………………………………………… 29 2-6-1. DNA mobility shift assay with a supercoiled pBluescript DNA substrate………………………………………………………………. 29 2-6-2. DNA mobility shift assay with an 80bp double-strand DNA (dsDNA)……………………………………………………………… 29 2-7. Yeast two hybrid…………………………………………………………….. 29 2-7-1 Yeast transformation for yeast two hybrid…………………………… 29 2-7-1-1 Preparation of yeast competent cells………………………… 29 2-7-1-2 Co-transformation of AD and BD yeast two hybrid plasmids. 30 2-7-2 Yeast two hybrid…………………………………………………........ 31 2-8. Plasmids list………………………………………………………………… 31 2-9. Buffer and medium list……………………………………………………… 32 CHAPTER 3: Results 3-1. Expression and purification of Rec27, Rec25, and Mug20………………… 34 3-1-1. Purification of Rec27 recombinant protein………………………….. 34 3-1-2. Purification of Rec25 recombinant protein………………………….. 35 3-1-3. Purification of Mug20 recombinant protein…………………………. 35 3-2. Rec27 is the key component in Rec27-Rec25-Mug20 complex……………. 36 3-3. The interaction relationship of Rec27, Rec25, and Mug20 by yeast two-hybrid analyses…………………………………………………………………... 38 3-4. Rec27 possesses a DNA binding activity…………………………………… 40 3-5. Basic amino acids clusters of Rec27 contribute to its DNA binding ability... 41 3-6. Expression and purification of Rec27-Rec25-Mug20 complex…………….. 42 3-7. Rec27-Rec25-Mug20 complex processes a DNA binding ability………….. 44 CHAPTER 4: Conclusion and Discussion 4-1 Summary of Key Findings…………………………………………………... 45 4-2 Discussion and Future Directions…………………………………………… 46 4-2-1 How do Rec27 DNA binding mutants affect homologous pairing and meiotic recombination?......................................................................... 46 4-2-2 Does Rec27-Rec25-Mug20 complex can physically interact Rec12 or Rec12 complex and affect the formation of meiotic DSBs?................. 48 4-2-3 The DNA binding property of Rec27 protein………………………… 48 4-2-4 Does Rec27 have sequence binding specificity?................................... 49 4-2-5 The choice of protein tags and their influence on protein functions…. 50 Figures…………………………………………………………………………. 51 References……………………………………………………………………... 83 Appendix A-1. List of N terminal tagging of Rec27, Rec25, and Mug20………………….. 88 A-2 Online prediction of potential DNA binding amino acids in Rec27 protein... 89 A-3 The prediction of nuclear localization signal (NLS) in Rec27……………… 90 A-4. Structure prediction of Rec27 wild type and 7A mutant…………………… 91 | |
dc.language.iso | zh-TW | |
dc.title | 探討Rec27、Rec25、Mug20在減數分裂中的同源染色體的配對以及同源重組中的角色與功能 | zh_TW |
dc.title | Investigating the Functional Roles of Rec27, Rec25, and Mug20 in Homologous Pairing and Meiotic Recombination | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李弘文(Hung-Wen Li),王廷方(Ting-Fang Wang),王中茹(Chung-Ju Wang) | |
dc.subject.keyword | Rec27,Rec25,Mug20,Rec12,線性單元,同源染色體配對,同源重組, | zh_TW |
dc.subject.keyword | Rec27,Rec25,Mug20,Rec12,linear elements,pairing,meiotic recombination, | en |
dc.relation.page | 92 | |
dc.identifier.doi | 10.6342/NTU201603454 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-21 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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