中文摘要
有絲分裂對於影響生物遺傳是重要的，依賴染色體上的著絲粒蛋白群(centromere)與著絲點蛋白群(kinetochore)行使穩定染色體結構及功能。在有絲分裂期間，透過著絲粒蛋白群吸引著絲點蛋白群結合並且提供紡錘絲微管(spindle microtubules)結合位。藉此提供姊妹染色體分離所需的雙向張力以維持染色體平均分配到每一個子細胞。染色體的纏繞基本單位──核小體(nucleosome)，一般H3核小體由組蛋白(histone) (H3, H4, H2A, H2B)及α-衛星DNA(α-satellite DNA)形成之八聚體，在著絲粒蛋白群由CENP-A取代H3形成CENP-A核小體，CENP-A是著絲粒蛋白的表觀遺傳標記。
在CENP-A核小體末端有一段特異性序列──CENP-B box，CENP-B box是提供CENP-B的DNA結合域(CENP-B DBD)特異性結合至CENP-A核小體。根據先前的研究，人類CENP-A核小體晶體結構(CENP-A 、H4、H2A、H2B
、α-衛星DNA) (PDB:3AN2)中，其中此區域之α-衛星DNA含有CENP-B box及CENP-A N端的結構尚未解析。在實驗中CENP-A 氨基端對於CENP-B在CENP-A核小體的穩定有關；DNA競爭實驗結果顯示CENP-B與CENP-A核小體結合較H3核小體穩定，但其詳細機制尚未明瞭。故本研究探討CENP-A 氨基端、CENP-B-CENP-B DBD、CENP-B box DNA三者的交互作用關係。
首先我們成功將CENP-B DBD及CENP-A N59(CENP-A N59，1-59個胺基酸)透過蛋白變性(denature)及再折疊(refolding)方法成功純化。利用EMSA證明CENP-B DBD和CENP-A N59均能與CENP-B box DNA透過DNA序列專一及DNA序列非專一的結合方式。
為探討CENP-B DBD、CENP-A N59之間的結合模式，首先以反向原態酸性聚丙烯醯胺膠體電泳確認成功移除結合在CENP-A N59大部分的大腸桿菌DNA，因為CENP-A N59淨電荷為正，大量表現時候容易黏附大腸桿菌DNA。再經由反向原態酸性聚丙烯醯胺膠體電泳探討CENP-B DBD及CENP-A N59，而其結果為兩者之間沒有明確的交互作用，故利用膠體過濾層析法探討生理環境下探討CENP-A N59與CENP-B DBD兩者似乎沒有明確的交互作用，進一步利用EMSA 探討CENP-A N59與CENP-B DBD-CENP-B box DNA複合物似乎有結合。推測CENP-A N59與CENP-B DBD需藉由CENP-B box DNA的參與。

Abstract
Mitosis effectively are critical for animal epigenetic. In order to assure DNA segregation accuracy, it should depend on centromere and kinetochore which locate on chromosome alpha-satellite DNA repeat to stabilize chromosome. During mitosis, centromere proteins recruit kinetochore proteins to offer a platform for spindle microtubule binding site. When spindle microtubules attach to each sister chromatids, it can offer pulling forces for bio-orientation tension across sister chromatids. Thereby, replicated DNA chromosome can be sorted into each daughter cell equally. The unite of chromosome, histone, include two copies of H2A, H2B, H3, H4, to wrap the DNA into 147 bps. Furthermore, each 147 bps DNA are condensed into high-order conformation to keep in nucleus.
CENP-A is characterized as variant of centromere-specific histone H3. Also, it is a specific epigenetic marker of centromere. According to previous study of human CENP-A nucleosome crystal structure (PDB:3AN2), it suggests that only the central 121 bps of the DNA are visible unlike H3 nucleosome. 13 bps from the end of DNA doesn’t be resolved in the CENP-A nucleosome crystal structure. Additionally, 17 bps from the end of CENP-A nucleosome, called CENP-B box, which is a specific region for CENP-B binding site in the CENP-A nucleosome. Not only in vivo but in vitro, CENP-A N-terminal is related to stability of CENP-B binding to CENP-A nucleosome. These study suggest that CENP-B bind more stable to CENP-A nucleosome than H3 nucleosome.
In order to investigate the interaction of CENP-B DNA binding protein (CENP-B DBD), CENP-A N59, CENP-B box. First, we successfully purify CENP-B DBD and CENP-A N59 by denature and renature method. Second, EMSA was applied to ensure CENP-B DBD whether renature successfully. Third, to investigate CENP-B DBD and CENP-A N59, we remove almost of contaminate DNA from CENP-A N59. Fourth, reverse acidic native gel was applied to observe the interaction of CENP-A N59 and CENP-B DBD. Fifth, size exclusion chromatography was applied to investigate the interaction of CENP-A N59 with CENP-B DBD.In the end, EMSA was applied to investigate the interaction of CENP-A N59 with complex CENP-B DBD-CENP-B box. According to reverse acidic native gel, size exclusion chromatography and EMSA, it supposed that CENP-B box may participate in the interaction of CENP-A N-terminal with CENP-B DBD.

目錄
目錄 I
中文摘要 III
Abstract IV
誌謝 V
第一章 前言 1
1.1 著絲點 (Centromere)、著絲粒 (Kinetochore) 蛋白群 1
1.2 H3核小體組成 2
1.3 CENP-A 核小體 3
1.4 CENP-A核小體與H3核小體比較 4
1.5 CENP-A在細胞週期的調節 6
1.6 CENP-A N端 7
1.7 CENP-B結構與功能 8
1.8 CENP-B與CENP-A核小體的交互作用 10
研究目標: 12
第二章 材料與方法 13
2.1 CENP-B DBD蛋白質在大腸桿菌表現與純化 13
2.1.1 CENP-B表現載體(expression vector)之製備 13
2.1.2 表現CENP-B DBD蛋白 13
2.1.3 透過尿素使CENP-B DBD變性(denature, d-CENP-B DBD) 14
2.1.4 CENP-B DBD的固定化金屬親和管柱層析(Immobilized metal-affinity chromatography，IMPAC) 14
2.1.5 f-CENP-B DBD透析作用 15
2.2 Electrophoretic mobility shift assay (EMSA, 電泳遷移率變動分析) 16
2.3 CENP-A N59 表現和純化 16
2.3.1 CENP-A N59表現載體(expression vector)之製備 16
2.3.2 表現CENP-A N59蛋白 17
2.3.3 變性d-CENP-A N59破菌 17
2.3.4變性d-CENP-A N59純化 17
2.3.5 d-CENP-A N59 膠體過濾層析法(gel filtration) 18
2.3.6 CENP-A N59 透析作用 (dialysis) 19
2.4 Benzonase 核酸分解酶 19
2.5 Thrombin protease 20
第三章.結果與討論 21
3.1 CENP-B的表現、純化及特性 21
3.1.1 CENP-B 的蛋白表現 21
3.1.2 d-CENP-B DBD及f-CENP-B DBD的純化 21
3.1.3 f-CENP-B DBD與CENP-B box DNA結合能力 22
3.1.4 f-CENP-B DBD二級結構 23
3.2 CENP-A N59的表現、純化及特性 23
3.2.1 CENP-A N59的表現 23
3.2.2 CENP-A N59純化 24
3.2.3 CENP-A N59上E.coli內生性DNA(contaminated DNA)去除測試 25
3.3 CENP-A N59與CENP-B box DNA結合能力 26
3.4 CENP-A N59與CENP-B DBD交互作用 26
3.5 CENP-A N59與CENP-B DBD-CENP-B box複合物交互作用 27
第四章 結論 30
圖片和表格 33
參考文獻 : 52

1. Darlington C.D. The external mechanics of chromosomes. Proc. R. Soc. Lond. B Biol. Sci. 1936;121:264–319. 1. The external mechanics of the chromosomes I—The scope of enquiry. Proceedings of the Royal Society of London. Series B - Biological Sciences, 1936. 121(823): p. 264.
2. Earnshaw, W.C., Discovering centromere proteins: from cold white hands to the A, B, C of CENPs. Nat Rev Mol Cell Biol, 2015. 16(7): p. 443-9.
3. Jia, L., S. Kim, and H. Yu, Tracking spindle checkpoint signals from kinetochores to APC/C. Trends Biochem Sci, 2013. 38(6): p. 302-11.
4. Simpson, R.T., Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones. Biochemistry, 1978. 17(25): p. 5524-5531.
5. McGinty, R.K. and S. Tan, Nucleosome structure and function. Chem Rev, 2015. 115(6): p. 2255-73.
6. Arents, G., et al., The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix. Proceedings of the National Academy of Sciences of the United States of America, 1991. 88(22): p. 10148-10152.
7. Arents, G. and E.N. Moudrianakis, The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization. Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(24): p. 11170-11174.
8. Luger, K., et al., Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature, 1997. 389: p. 251.
9. Yoda, K., et al., Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proceedings of the National Academy of Sciences, 2000. 97(13): p. 7266.
10. Tanaka, Y., et al., Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences. J Biol Chem, 2005. 280(50): p. 41609-18.
11. Camahort, R., et al., Cse4 is Part of an Octameric Nucleosome in Budding Yeast. Molecular cell, 2009. 35(6): p. 794-805.
12. Fujita, R., et al., Stable complex formation of CENP-B with the CENP-A nucleosome. Nucleic Acids Res, 2015. 43(10): p. 4909-22.
13. Barnhart, M.C., et al., HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore. The Journal of Cell Biology, 2011. 194(2): p. 229-243.
14. Guse, A., et al., In vitro centromere and kinetochore assembly on defined chromatin templates. Nature, 2011. 477(7364): p. 354-358.
15. Mendiburo, M.J., et al., Drosophila CENH3 Is Sufficient for Centromere Formation. Science, 2011. 334(6056): p. 686.
16. Tachiwana, H., et al., Crystal structure of the human centromeric nucleosome containing CENP-A. Nature, 2011. 476(7359): p. 232-5.
17. Allshire, R.C. and G.H. Karpen, Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nature Reviews Genetics, 2008. 9: p. 923.
18. Cheeseman, I.M. and A. Desai, Molecular architecture of the kinetochore–microtubule interface. Nature Reviews Molecular Cell Biology, 2008. 9: p. 33.
19. Earnshaw, W.C. and N. Rothfield, Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma, 1985. 91(3-4): p. 313-21.
20. Henikoff, S., et al., Heterochromatic deposition of centromeric histone H3-like proteins. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(2): p. 716-721.
21. Conde e Silva, N., et al., CENP-A-containing Nucleosomes: Easier Disassembly versus Exclusive Centromeric Localization. Journal of Molecular Biology, 2007. 370(3): p. 555-573.
22. Panchenko, T., et al., Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical termini. Proc Natl Acad Sci U S A, 2011. 108(40): p. 16588-93.
23. McKinley, K.L., et al., The CENP-L-N complex forms a critical node in an integrated meshwork of interactions at the centromere-kinetochore interface. Molecular cell, 2015. 60(6): p. 886-898.
24. Carroll, C.W., et al., Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N. Nat Cell Biol, 2009. 11(7): p. 896-902.
25. Nagpal, H., et al., Dynamic changes in CCAN organization through CENP-C during cell-cycle progression. Molecular Biology of the Cell, 2015. 26(21): p. 3768-3776.
26. Przewloka, M.R., et al., CENP-C is a structural platform for kinetochore assembly. Curr Biol, 2011. 21(5): p. 399-405.
27. Fachinetti, D., et al., A two-step mechanism for epigenetic specification of centromere identity and function. Nat Cell Biol, 2013. 15(9): p. 1056-66.
28. Sekulic, N., et al., The structure of (CENP-A-H4)(2) reveals physical features that mark centromeres. Nature, 2010. 467(7313): p. 347-51.
29. Black, B.E., et al., Structural determinants for generating centromeric chromatin. Nature, 2004. 430: p. 578.
30. Shelby, R.D., K. Monier, and K.F. Sullivan, Chromatin Assembly at Kinetochores Is Uncoupled from DNA Replication. The Journal of Cell Biology, 2000. 151(5): p. 1113.
31. Nechemia-Arbely, Y., D. Fachinetti, and D.W. Cleveland, Replicating centromeric chromatin: spatial and temporal control of CENP-A assembly. Experimental cell research, 2012. 318(12): p. 1353-1360.
32. Dunleavy, E.M., et al., HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres. Cell, 2009. 137(3): p. 485-97.
33. Foltz, D.R., et al., Centromere specific assembly of CENP-A nucleosomes is mediated by HJURP. Cell, 2009. 137(3): p. 472-484.
34. Hu, H., et al., Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP. Genes Dev, 2011. 25(9): p. 901-6.
35. Yu, Z., et al., Dynamic phosphorylation of CENP-A at Ser68 orchestrates its cell-cycle-dependent deposition at centromeres. Dev Cell, 2015. 32(1): p. 68-81.
36. Hasson, D., et al., The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat Struct Mol Biol, 2013. 20(6): p. 687-95.
37. Roulland, Y., et al., The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity. Mol Cell, 2016. 63(4): p. 674-685.
38. Tsunaka, Y., et al., Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle. Nucleic Acids Research, 2005. 33(10): p. 3424-3434.
39. Tachiwana, H., et al., Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T. Proc Natl Acad Sci U S A, 2010. 107(23): p. 10454-9.
40. Iwahara, J., et al., A helix-turn-helix structure unit in human centromere protein B (CENP-B). Vol. 17. 1998. 827-37.
41. A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity. The Journal of Cell Biology, 1992. 119(6): p. 1413-1427.
42. Tawaramoto, M.S., et al., Crystal structure of the human centromere protein B (CENP-B) dimerization domain at 1.65-A resolution. J Biol Chem, 2003. 278(51): p. 51454-61.
43. Kitagawa, K., et al., Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle. Vol. 15. 1995. 1602-12.
44. Wang, H., E. Hartswood, and D.J. Finnegan, Pogo transposase contains a putative helix-turn-helix DNA binding domain that recognises a 12 bp sequence within the terminal inverted repeats. Nucleic Acids Research, 1999. 27(2): p. 455-461.
45. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. The Journal of Cell Biology, 1989. 109(5): p. 1963-1973.
46. Tanaka, Y., et al., Crystal structure of the CENP-B protein–DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA. The EMBO Journal, 2001. 20(23): p. 6612-6618.
47. Okada, T., et al., CENP-B controls centromere formation depending on the chromatin context. Cell, 2007. 131(7): p. 1287-300.
48. Fachinetti, D., et al., DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function. Dev Cell, 2015. 33(3): p. 314-27.
49. Tachiwana, H., et al., Nap1 regulates proper CENP-B binding to nucleosomes. Nucleic Acids Res, 2013. 41(5): p. 2869-80.
50. Micsonai, A., et al., Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proceedings of the National Academy of Sciences of the United States of America, 2015. 112(24): p. E3095-E3103.