臺灣博碩士論文加值系統

　　聯會複合體 (synaptonemal complex, SC)為減數分裂專一性之染色體特化構造，是由兩側的軸元細絲(axial element)及中央元素 (central element)所構成三層蛋白質結構。在減數分裂的前期，聯會複合體負責聯會(synapse)來自父母雙方的同源染色體，促進同源DNA重組 (homologous DNA recombination)發生於同源染色體之間，此過程除了產生新的遺傳變異之外，機制上也確保成對的同源染色體在第一次減數分裂時能平均地分離。Red1與Hop1是出芽酵母菌 (Saccharomyces cerevisiae) 軸元細絲上的結構蛋白，當Spo11催化DNA產生雙股斷裂後，Red1協助活化Mec1與Tel1蛋白激酶將Hop1進行磷酸化，磷酸化的Hop1接著活化Mek1蛋白激酶，而活化的Mek1則會磷酸化Rad54的第132個胺基酸蘇胺酸 (threonine)，進而降低其與DNA重組酶Rad51的交互作用，這樣的過程會減緩同源DNA重組發生於來自親代單方的姐妹染色分體 (sister chromatids)之間，間接地增加同源DNA重組發生於同源染色體間的機會。因此red1/mek1/hop1的突變株，同源染色體間DNA重組作用發生機會下降，同源染色體不能平均分離，嚴重降低孢子的存活率。為了能偵測體內Mek1的激酶活性，我們設計生產了抗磷酸化Rad54-T132的抗體，並將之應用於檢測不同酵母菌突變株中Rad54磷酸化的情形，藉此了解Mek1活化的相關機制。我發現半顯性活性 (semi-dominant active)的GST-Mek1蛋白，其激酶活性活化與否由細胞內Hop1的第318個胺基酸蘇胺酸318 (T318)被磷酸化的量而定，而且Mek1可能是經由其FHA (forkhead-associated) 功能區的第51個胺基酸精胺酸 (arginine)與第88個胺基酸賴胺酸 (lysine)與磷酸化的Hop1-T318進行結合，這些結果確認Mek1的活化需經由磷酸化的Hop1。最後我利用酵母菌雙雜合系統發現Mek1的FHA功能區可能也與小泛素 (small ubiquitin-like modifier, SUMO)聚合鏈有交互作用，但此作用並不影響Mek1的活化。

　　The synaptonemal complex (SC) is a meiosis-specific tripartite protein structure consisting of two axial elements and one central element. The SC is thought to promote homologous DNA recombination between homologous chromosomes during the meiotic prophase. Interhomolog recombination not only generates genetic diversity in gametes but also ensures proper segregation of homologous chromosomes during the first meiotic nuclear division (MI). In budding yeast Saccharomyces cerevisiae, Red1 and Hop1 are meiosis-specific chromosomal proteins that localize to axial elements. In response to Spo11-induced DNA double-strand breaks (DSBs), Red1 activates Mec1/Tel1 DNA damage checkpoint kinases. Mec1/Tel1 phosphorylate the threonine 318 (T318) residue of Hop1, which in turn leads to activation of Mek1 protein kinase. Activated Mek1 then phosphorylates the threonine 132 (T132) residue of Rad54, an accessory factor of RecA-like recombinase Rad51. Compared to nonphosphorylated Rad54, phosphorylated Rad54-T132 exhibits a much weaker physical and functional interaction with Rad51. As a result, the rate of homologous DNA recombination between the two sister chromatins (i.e., intersister recombination) is greatly reduced. The red1/mek1/hop1 mutants all exhibit higher levels of intersister recombination and reduced levels of interhomolog recombination, and therefore they produce very few viable spores due to mis-segregation of homologous chromosomes at MI. In the present study, I first generated antibodies against phosphorylated Rad54-T132 and then applied these antibodies to study the activation mechanism of Mek1. My results revealed that phosphorylated Hop1-T318 is required for activation of GST-Mek1, a semi-dominant active form of Mek1. GST-Mek1 is able to stabilize the phosphorylated Hop1-T318. Two positively charged amino acid residues, arginine 51 and lysine 88 in the forkhead-associated domain (FHA domain) of Mek1, likely are responsible for binding to phosphorylated Hop1-T318. Finally, I showed that Mek1 can also interact with the polymeric chain of small ubiquitin-like modifier (SUMO), but that novel interaction is not required for Mek1 activation.

中文摘要 ..................................................I
Abstract..................................................II
目錄.....................................................III
縮寫.......................................................V
第一章前言.................................................1
1.1 同源染色體DNA重組對減數分裂的重要性....................1
1.2 酵母菌減數分裂過程中DNA重組的機制......................1
1.3 DNA破損檢查點..........................................3
1.4 聯會複合體的形成機制...................................3
1.5 聯會複合體的生化功能...................................4
1.6 Mek1幫助同源染色體的重組...............................6
1.7 GST-MEK1為半顯性功能增加的對偶基因.....................6
第二章 實驗方法與材料......................................8
2.1 酵母菌基因轉型.........................................8
2.2 酵母菌減數分裂週期同步化...............................8
2.3 三氯乙酸蛋白質沉澱法...................................9
2.4 蛋白質電泳與西方點墨法.................................9
2.5 細胞核散灑............................................10
2.6 染色體免疫螢光染色....................................11
2.7 酵母菌雙雜合系統......................................12
2.8 酵母菌品系............................................13
2.9 質體..................................................14
2.10 實驗培養基...........................................15
2.11 實驗溶液配方.........................................16
第三章 結果...............................................18
3.1 利用抗磷酸化Rad54-T132之抗體分析Mek1的激酶活性........18
3.2 GST-Mek1的活化需要磷酸化的Hop1-T318...................19
3.3 mek1-R51A與mek1-K88A突變株影響Rad54的磷酸化...........20
3.4 利用酵母菌雙雜合系統分析Mek1與小泛素蛋白之間的交互作用22
3.5 分析mek1-SIM突變株對Mek1活性的影響....................23
3.6 不同標籤對於Mek1突變蛋白的影響........................23
第四章 討論...............................................25
4.1 以抗磷酸化Rad54-T132之抗體分析Mek1的激酶活性..........25
4.2 GST-Mek1的活化需要磷酸化的Hop1-T318...................25
4.3 在減數分裂過程中Mek1可能與Smt3聚合鏈結合..............26
第五章 參考文獻...........................................28
第六章 結果與圖表.........................................31
表格一、利用酵母菌雙雜合系統分析Mek1與SUMO之間的交互作用..31
表格二、以四分體孢子分離分析酵母菌之孢子存活率............32
圖一、分析不同突變株之Rad54 磷酸化情形....................33
圖二、利用Rad54的磷酸化分析GST-Mek1蛋白在mek1與hop1T318A突變株中活化的情形............................................34
圖三、分析GST-Mek1對粗絲期檢查點的影響....................35
圖四、分析不同mek1突變株之Rad54磷酸化情形.................36
圖五、偵測Mek1在不同突變株中結合到染色體的情形............37
第七章 附錄...............................................38
圖一、減數分裂過程中DNA的重組機制.........................38
圖二、分析Mek1胺基酸序列..................................39

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