根據長庚醫院的統計資料顯示，乳癌在女性癌症發生率中位居第二，僅次於子宮頸癌。蛋白質BRCA1(乳癌相關蛋白質1)，是一種乳癌抑制者。先前的研究發現，BRCA1會與BARD1(與BRCA1有交互作用的蛋白質)形成異源二聚體，並且具有E3泛素連接酶的活性。他們在細胞週期中扮演重要的角色，像是參與脫氧核糖核酸的損傷修復及微管的形成。研究發現knockdown BRCA1會導致中心體不正常的增生。同時也發現BRCA1在G2 phase的活性較高，但並不知道是什麼因子在調控著。直到2014年日本團隊發現了新的結合機制，透過質譜儀分析找出能夠與BRCA1/BARD1產生交互作用的蛋白質OLA1。先前的文獻在乳腺癌細胞株中發現OLA1的突變(E168Q) 且不會與BRCA1有交互作用。同時也發現knockdown OLA1也會導致中心體不正常的增生。然而，OLA1與BRCA1/BARD1所形成複合體的結構及功能還是一個未知。
因此，我們主要想研究OLA1與BRCA1/BARD1之間的交互作用。在研究中，我們利用了核磁共振光譜學觀察發現OLA1與BARD1 BRCT在ATP 狀態下有交互作用，同時也利用點突變及三磷酸腺苷水解活性的實驗找到OLA1在BARD1 BRCT上的可能結合位點。未來可以藉由X光晶體來分析他們的複合體結構，進而應用在癌症生物學上。

According to the statistics from the Chang Gung Memorial Hospital, the incidence of breast cancer is the second highest in women, only next to cervical cancer. The protein BRCA1 (Breast cancer-associated gene 1), which is a breast tumor suppressor, interacts with the protein BARD1 (BRCA1-associated RING domain protein), resulting in the formation of a heterodimeric complex that has E3 ubiquitin ligase activity and plays a central role in cell cycle checkpoint control, DNA repair, and microtubule (MT) aster formation. The protein OLA1 (Obg-like ATPase 1) is a new binding partner of BRCA1/BARD1. A previous study found that OLA1 mutation (E168Q), which was observed in breast cancer cell line, failed to bind BRCA1. However, the structural basis and functional consequences of the interaction of OLA1 with BRCA1/BARD1 are unknown yet.
Our goal is to understand the molecular basis of the interaction of OLA1 with BRCA1/BARD1. We used NMR TROSY-HSQC experiments to detect interaction of OLA1 and BRCA1/BARD1 in the apo and ATP state. We used ATPase assays in combination with site-directed mutagenesis to demonstrate a potential OLA1 binding site on BARD1 BRCT. Structural information derived from this study provides a new insight to fundamental as well as breast cancer biology.

中文摘要 1
ABSTRACT 2
Abbreviations 3
Chapter 1 Introduction 4
Chapter 2. Materials and methods 9
Chapter 3. Results 19
Chapter 4. Discussion & Conclusion 24
Chapter 5. Perspective 28
REFERENCE 29
Appendix 62

1. Fox, D.L.T., I. Rajagopal, P. Brzovic, P. S. Stenkamp, R. E. Klevit, R. E., Crystal structure of the BARD1 ankyrin repeat domain and its functional consequences. Journal of Biological Chemistry, 2008. 283(30): p. 21179-21186.
2. Roy, R., J. Chun, and S.N. Powell, BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer, 2012. 12(1): p. 68-78.
3. Sankaran, S.S., L. M. Groen, A. C. Ko, M. J. Parvin, J. D., Centrosomal microtubule nucleation activity is inhibited by BRCA1-dependent ubiquitination. Molecular and Cellular Biology, 2005. 25(19): p. 8656-8668.
4. Xiaoling Xu, Z.W., Steven P. Linke, Cuiling Li, Jessica Gotay, Xin-Wei Wang, Curtis C. Harris, Thomas Ried, and Chu-Xia Deng, Centrosome Amplification and a Defective G2–M Cell Cycle Checkpoint Induce Genetic Instability in BRCA1 Exon 11 Isoform–Deficient Cells. Molecular Cell,1999. 3: p. 389–395.
5. Christensen, D.E., P.S. Brzovic, and R.E. Klevit, E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol, 2007. 14(10): p. 941-8.
6. Peter S. Brzovic, P.R., David W. Hoyt, Mary-Claire King and Rachel E. Klevit, Structure of a BRCA1–BARD1 heterodimeric RING–RING complex. Nature structural biology, 2001. 8: p. 98195-7742.
7. Hashizume, R.F., M. Maeda, I. Nishikawa, H. Oyake, D. Yabuki, Y. Ogata, H. Ohta, T., The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. J Biol Chem, 2001. 276(18): p. 14537-40.
8. Deng, C.-X., Roles of BRCA1 in centrosome duplication. Nature, 2002. 21: p. 6222 – 6227.
9. To Hoa Thai, F.D., Julia Tsou Tsan, Ying Jin, Anne Phung. Mutations in the BRCA1-associated RING domain (BARD1) gene in primary breast, ovarian and uterine cancers. Human Molecular Genetics, 1998. 7: p. 195–202.
10. To Hoa Thai, F.D., Julia Tsou Tsan, Ying Jin, Anne Phung, Monique A. Spillman, Hillary F. Massa, Carolyn Y. Muller, Raheela Ashfaq, J. Michael Mathis, David S. Miller, Barbara J. Trask, Richard Baer and Anne M. Bowcock, Mutations in the BRCA1-associated RING domain (BARD1) gene in primary breast, ovarian and uterine cancers. Human Molecular Genetics, 1998. 7: p. 195–202.
11. Clark, S.L.R., A. M. Snyder, R. R. Hankins, G. D. Boehning, D., Structure-Function Of The Tumor Suppressor BRCA1. Comput Struct Biotechnol J, 2012. 1(1).
12. Gabriel Birrane, A.K.V., Aditi Soni, and John A. A. Ladias, Crystal Structure of the BARD1 BRCT Domains. Biochemistry, 2007. 46: p. 7706-7712.
13. Isaac A. Manke, D.M.L., Anhco Nguyen, Michael B. Yaffe, BRCT Repeats As Phosphopeptide-Binding Modules Involved in Protein Targeting. Science, 2003. 302: p. 636-639.
14. Feng, B., et al., Structural and functional insights into the mode of action of a universally conserved Obg GTPase. PLoS Biol, 2014. 12(5): p. e1001866.
15. Koller-Eichhorn, R.M., T. Gail, R. Wittinghofer, A. Kostrewa, D. Kutay, U. Kambach, C., Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins. J Biol Chem, 2007. 282(27): p. 19928-37.
16. Hannemann, L.S., I. Ba, Q.MacInnes, K. Drepper, F. Warscheid, B. Koch, H. G., Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1. Antioxid Redox Signal, 2015. 24(3): p. 141-56.
17. Detlef D. Leipe, Y.I.W., Eugene V. Koonin and L. Aravind, Classification and Evolution of P-loop GTPases and Related ATPases. J. Mol. Biol, 2002. 317: p. 41-72.
18. Matsuzawa, A.K., S. Nakayama, M. Mochiduki, H. Wei, L. Shimaoka, T.Furukawa, Y. Kato, K. Shibata, S. Yasui, A. Ishioka, C. Chiba, N., The BRCA1/BARD1-interacting protein OLA1 functions in centrosome regulation. Mol Cell, 2014. 53(1): p. 101-14.
19. German A. Pihan, J.W., Yening Zhou, and Stephen J. Doxsey, Centrosome Abnormalities and Chromosome Instability Occur Together in Pre-invasive Carcinomas. CANCER RESEARCH, 2003. 63: p. 1398–1404.
20. Washington, U.o., Structural Characterization and Protein Interactions
Involving the C-terminal Domains of the Breast Cancer Susceptibility Protein,
BRCA1, and its Constitutive Partner, BARD1. 2008: p. 48106-1346.
21. Daniel G Gibson, L.Y., Ray-Yuan Chuang, J Craig Venter, Clyde A Hutchison & Hamilton O Smith, Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 2009. 6: p. 343 - 345.
22. Christensen, D.E., P.S. Brzovic, and R.E. Klevit, E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol, 2007. 14(10): p. 941-8.