黏著蛋白(cohesin)在許多細胞過程中扮演重要角色，其中包含DNA雙股斷裂損傷(double-stranded break, DSB)修復。先前研究顯示，DNA DSB會誘導黏著蛋白聚集到損傷部位(Unal et al. 2004)，並促進細胞利用姊妹染色體做同源性重組修復(Homologous recombination repair, HRR) (Sjögren and Nasmyth 2001)。MCD1為cohesin complex中最大的蛋白，MCD1若出現基因缺失會影響細胞利用姊妹染色體做HRR的效率(Birkenbihl and Subramani 1992)。
然而，MCD1對於細胞使用同條染色體(intrachromosomal)上具同源性的模板做HRR的影響尚未完全證實。本次研究，我們想知道MCD1是否也會影響細胞使用同條染色體做HRR。在出芽酵母菌中，我們利用galactose-inducible HO endonuclease誘導染色體在特殊位點上產生DSB，DSB被設計成能透過兩個同源供體(donor)之一做修復，一個在同條染色體上，另一個則是在其他染色體上並作為參考使用。通過觀察修復後的DNA序列，我們可以評估兩個供體之間的相對使用情形。接著，我們使用auxin-inducible degron (AID) system將細胞中的MCD1蛋白降解，以研究MCD1對於同條染色體基因轉換的生理作用。
實驗結果顯示，MCD1能在加入生長素後30分鐘內被降解。另外，我們意外發現誘導產生的DSB似乎會上調MCD1的表現量，但添加生長素仍足以誘導 MCD1降解完全。通過分析修復後的DNA序列，我們發現當MCD1降解完全時，同條染色體上的同源供體的相對使用率會降低，表明MCD1會促進同條染色體上的基因轉換。

Cohesin plays important roles in various cellular processes, including DNA double-stranded break (DSB) repair. Previous studies indicated that DNA DSBs induced the recruitment of cohesin to the damage site (Unal et al. 2004) and promoted homologous recombination repair (HRR) with sister chromatid (Sjögren and Nasmyth 2001). MCD1 is the largest subunit of cohesin complex. Deletion of the MCD1 gene affects the efficiency of DNA HRR with sister chromatid (Birkenbihl and Subramani 1992).
However, the effect of MCD1 on HRR using intrachromosomal homologous template is not being carefully examined yet. In this study, we wanted to investigate whether MCD1 affects HRR through intrachromosomal gene conversion. We utilized galactose-inducible HO endonuclease to induce a site-specific DSB in budding yeast. The DSB was designed to be able to be repaired via one of the two homologous donors, one of which is intrachromosomal and the other is on another chromosome and serves as a reference. By examining the sequence of repaired DNA region, we can estimate the relative usage of the two donors. We then established an auxin-inducible degron (AID) system to be able to degrade MCD1 to study the physiological role of MCD1 in intrachromosomal gene conversion.
The results showed that MCD1 can be degraded in 30 minutes after addition of auxin. We accidentally found that the induced DSB seemed to increase the expression of MCD1 but addition of auxin is still sufficient to induce complete depletion of MCD1. By analyzing the repaired DNA sequence, we found that when MCD1 was depleted, the relative usage of intrachromosomal homologous donor was decreased, suggesting that MCD1 promotes intrachromosomal gene conversion.

《中文摘要》...1
《ABSTRACT》...2
目錄...3
一、正文...5
1. 前言介紹...5
1.1 DNA雙股斷裂損傷修復...5
1.2 同源性重組修復...5
1.3 黏著蛋白的生理作用與重要性...6
1.4 MCD1的重要性與對HRR的影響...6
1.5 同條染色體基因轉換...7
2. 研究目標...8
2.1 實驗假說...8
2.2 實驗目的...8
3. 實驗器材、方法...9
3.1 Galactose-inducible HO (GAL-HO) system...9
3.2 Auxin-inducible degron (AID) system...9
圖1. 利用Auxin-inducible degron (AID) system降解MCD1示意圖...10
3.3 酵母菌株、質體...11
3.4 酵母菌培養液與藥品...11
3.5 酵母菌樣本收集...12
3.6 西方墨點法(Western blotting)...12
3.7 凝膠電泳法(agarose gel electrophoresis)...12
4. 實驗結果...13
4.1 建構能被誘導降解MCD1的酵母菌株...13
4.1.1 使用能觀察修復模板使用狀況的酵母菌株...13
圖2. GAL-HO系統的運作與細胞修復狀況觀察方法...15
4.1.2 建構含AID系統的酵母菌...17
圖3. 建構出含有AID系統的酵母菌株...19
圖4. 測試生長素IAA對YCSL001、YAID001生長速度的早期影響...20
4.2 MCD1能在30分鐘內被誘導降解...22
圖5. 測試MCD1是否能被IAA誘導降解...23
4.3 DSB會影響MCD1的表現量...24
圖6. 測試DSB對MCD1的表現量影響...25
4.4 MCD1降解效果不受DSB影響...26
圖7. MCD1的降解效果不受DSB影響...27
4.5 MCD1降解讓同條染色體做修復模板的比例下降...29
圖8. MCD1降解讓同條染色體(intrachromosomal)做HRR的比例下降...31
5. 總結、討論...32
5.1 MCD1會影響細胞利用同條染色體做HRR...32
5.2 建構YAID001過程的困境...33
5.3 AID系統在實驗中的優勢與劣勢...33
5.4 細胞週期狀態不同、生長狀態不同...35
6. 未來的實驗方向...36
6.1 蛋白定量、抽取方式優化...36
6.2 分析修復模板比率方法...36
6.3 考慮MCD1在不同生長週期時的表現量...37
表格1. 本次研究中所使用的酵母菌株...38
表格2. 本次研究中所使用的質體...38
表格3. 本次研究中所使用的primer...39
補充圖1. 建構YAID001 過程中使用的質體...41
二、參考文獻...42
《ACKNOWLEDGMENT》...44

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