BRCA1 (Breast cancer-associated susceptibility gene 1)是一種腫瘤抑制蛋白，而BRCA1被發現與家族遺傳性的乳癌與卵巢癌息息相關．在先前的研究中發現BRCA1會與BARD 1 (BRCA1-associated RING domain protein 1)形成二元體，進而參與細胞之中的調控，像是DNA修復、轉錄調控、中心體調控等．中心體是動物細胞中主要生成微管的地方，在細胞進行有絲分裂的過程中，中心體會被複製成一對，而此複製過程是經由非常精密的調控，當調控出現了缺陷，會導致中心體被複製成大於兩個的不正常增生，此種不正常的增生會與癌細胞生成有關。
BRCA1/BARD1和OLA1會藉由BRCA1/BARD1上N端的RING區域與BARD 1上Ｃ端的BRCT區域互相結合，並形成複合體．而這個複合體在細胞分裂的過程之中會座落於中心體內，而在先前的研究中也發現到了如果用細胞技術將OLA1或者是BRCA1去除會導致似程度的中心體不正常增生．而此OLA1與BRCA1/BARD1在細胞分裂中是如何調控中心體的還是未知。
所以在此篇研究中我們想探討OLA1與BRCA1/BARD1這個複合體的結構與功能．我們實驗室先前的研究發現到了，在體外實驗BRCA1/BARD1的RING區域會增強OLA1水解ATP的能力．為了近一步了解RING是如何增加OLA1水解ATP的能力，我們利用了生化與結構的分析．結果發現到RING是扮演著ATPase-activating protein來增加OLA1水解ATP的能力．而我們也進一步的搜尋在乳癌病人中所發現到在BRCA1的RING區域中許多突變點，從這些突變點中進而搜尋會產生中心體不正常的增生，我們發現到了一個突變點會減少RING幫助OLA1水解ATP的能力．綜合我們的研究，我們提供了一個BRCA1的突變與中心體不正常增生在分子層面上的解釋。

BRCA1 (Breast cancer-associated susceptibility gene 1) is a tumor suppressor that is associated with familial breast and ovarian cancers. BRCA1 binds BRCA1-associated RING domain protein 1 (BARD 1) to form a heterodimer, which participates in cellular processes including DNA damage repair, transcriptional regulation and centrosome regulation. Centrosomes are the major microtubule nucleation centers in animal cells. Duplication of the centrosome is finely controlled. Defects in centrosome regulation lead to centrosome amplification, which is linked to cancer. BRCA1/BARD1 binds to OLA1 (Obg-like ATPase 1) via BRCA1/BARD1 N-terminal RING (Really Interesting New Gene) domain and BARD1 C-terminal BRCT (BRCA1 C Terminus) domain. BRCA1/BARD1 and OLA1 are found to colocalize in the centrosome during cell division. It has been observed knockdown of OLA1 and/or BRCA1 results in a similar degree of centrosome amplification. How BRCA1/BARD1 and OLA1 participate in centrosome regulation, however, is still unclear. In this study, we investigated the structure and function relationship of OLA1- BRCA1/BARD1 RING complex. We discovered the RING domain of BRCA1/BARD1 increases the ATP hydrolysis activity of OLA1 in vitro. To further understand the underlining mechanism, we used biochemical and structural analyses to reveal that RING biochemically acts as an ATPase-activating protein to increase OLA1 ATPase activity. Next, we characterized BRCA1 RING domain mutations that lead to centrosome amplification during cell division. Indeed, one of the mutations decreases the OLA1 ATPase activity. Our work provides a molecular explanation for one BRCA1 mutation in centrosome amplification.

中文摘要 i
Abstract ii
致謝 iii
Content iv
Abbreviation 1
Chapter 1. Introduction 3
1.1 BRCA1/BARD1 3
1.2 Centrosome 4
1.3 Human Obg-like ATPase 1 (hOLA1) 5
1.4 Specific aim 7
Chapter 2. Materials and Methods 8
2.1 Plasmid construct design 8
2.2 Protein expression and purification 9
2.3 ATPase activity assay 12
2.4 Isothermal titration calorimetry (ITC) 12
2.5 Circular dichroism spectroscopy 13
2.6 Size exclusion chromatography 13
Chapter 3. Results 15
3.1 ATPase activity of artificial RING-OLA1 fusion was more active than OLA1-RING fusion. 15
3.2 The Michaelis-Menten analysis revealed that RING increased the kcat of OLA1. 15
3.3 OLA1 and RING-OLA1 had similar ATP and ADP binding affinities. 16
3.4 BRCT and RING displayed synergistic stimulation in OLA1. 17
3.5 RING(I21V) reduced the RING-stimulated ATPase activity of OLA1 17
3.6 The secondary structure content and melting temperature of RING-OLA1 and its mutants were similar. 18
3.7 The shape of BRCA1/BARD1 RING and mutants are similar 19
Chapter 4. Conclusion & Discussion 21
4.1 Conclusion 21
4.2 Discussion 22
Reference 24
Figures 27

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