臺灣博碩士論文加值系統

RSC複合物為一至少包含十五個蛋白質成員的複合體，利用ATP水解的能力來調節DNA與組蛋白之間的交互作用和核小體的移動情形，使原本包覆完整的DNA調控片段暴露出來以利轉錄因子結合，達到在酵母菌的基因體中調節基因表現的作用。Htl1p是RSC複合物中的蛋白質成員之ㄧ，且Htl1p的刪除會導致整個RSC複合體的不穩定。為了更進一步的了解為何將Htl1p刪除會導致此一不穩定的情形產生，便以TAP純化分析法來比較在RSC2-TAP與htl1刪除株,RH2-TAP, 純化出來的RSC複合物成員有何不同，從實驗結果觀察到Rsc58p似乎在htl1刪除株中較不穩定，容易被分解。而在Htl1p的突變蛋白(Htl1-7p)的存在下，利用TAP純化出的蛋白質成員與RSC2-TAP和RH2-TAP所純化出來的有明顯的不同。利用免疫分析來檢測，觀察到了在Htl1-7p存在下，利用TAP純化出來的Rsc8p與Rsc58p相對量明顯的減少。另外經由RSC複合物其他成員，Rsc58p與Htl1p，也可以成功的純化出RSC複合物成員。接著利用TAP來分析C端刪除的rsc8突變株對於RSC複合物的完整性有何影響，觀察到經由RSC2-TAP和在RH2-TAP酵母菌背景下製造的C端刪除片段的rsc8突變株所純化下來的蛋白質成員沒有明顯的不同。而我們實驗室之前的結果指出，在htl1刪除的情形之下可能會影響到Rsc8p與RSC複合物之間的穩定結合。為了更進一步的了解Rsc8p與RSC複合物之間的交互作用，便設計了一段融合蛋白SLR，SLR為利用Hsc70中的GGMP重複片段當連接子，將Sth1p與Rsc8p聯結起來。但在Htl1p不存在的情形下，帶有SLR的酵母菌株有致死現象產生; 但SLR可以拯救經由Tet-off 調控系統所抑制的RSC8表現所造成的致死現象，暗示SLR為一具有功能性的蛋白。根據以上結果可以更進一步的確認Htl1p為穩定RSC
複合物的重要因子。

The RSC complex, which consists of at least fifteen proteins, alters the histone-DNA interactions and nucleosome mobility by ATP hydrolysis, allowing transcription factors accessing to chromatin for a broad range of gene regulation in yeast genome. Htl1p is a component of RSC complex. In the absence of Htl1p, the integrity of RSC complex is disturbed. To better understand the role of Htl1p in the RSC complex, here we examine the components of RSC complex by tandem affinity purification and compare the differences of RSC components purified from RSC2-TAP, which chromosome RSC2 gene was fused with a TAP-tag at the c-terminus and its htl1 deletion derivative RH2-TAP. We observed that the Rsc58p seems to be unstable in RH2-TAP. Moreover, in the presence of Htl1p mutant protein (Htl1-7), the protein profile is different from that of RSC2-TAP and RH2-TAP. By immunoblot analysis, we observed that the relative quantity of Rsc58p and Rsc8p in the presence of Htl1-7 protein is decreased in the purified protein profile. Htl1p and Rsc58p associated proteins were also purified by tandem affinity purification. They are essential RSC components. However, in comparison of the protein profiles purified by the rsc8 mutants derived from RSC2-TAP and RH2-TAP, there’s no difference among them. Our preliminary data showed that deletion of htl1 affects the interaction between Rsc8p and RSC complex. To further understand the interaction between Rsc8p and RSC complex, we design a fusion protein SLR, using a linker containing the GGMP repeats of Hsc70 to connect Sth1p and Rsc8p. Yeast cells contain SLR with deletion of htl1 failed to survive. But SLR could rescue the lethal phenotype caused by down-regulation of RSC8 expression in tet-off system, indicating that SLR may replace the function of both RSC8 and STH1. Overall speaking, above results further confirm that Htl1p may be crucial for the stability of
RSC complex.

英文摘要………………………..………………………………………Ⅰ
中文摘要…………………..……………………………………………Ⅱ
目錄……………..………………………………………………………Ⅲ
緒論………………………………………………………………………1
材料與方法………………………………………………………………9
結果………………………………………………..……………………17
討論……………………..………………………………………………27
參考文獻………………………………………………………………37
圖表……………………………………………………………………43
附圖……………………………………………………………………69

參考文獻
1. Becker, P.B. and W. Horz, ATP-dependent nucleosome remodeling. Annu Rev Biochem, 2002. 71: p. 247-73.
2. Saha, A., J. Wittmeyer, and B.R. Cairns, Chromatin remodeling through directional DNA translocation from an internal nucleosomal site. Nat Struct Mol Biol, 2005. 12(9): p. 747-55.
3. Lia, G., et al., Direct observation of DNA distortion by the RSC complex. Mol Cell, 2006. 21(3): p. 417-25.
4. Mohrmann, L. and C.P. Verrijzer, Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes. Biochim Biophys Acta, 2005. 1681(2-3): p. 59-73.
5. Cairns, B.R., et al., RSC, an essential, abundant chromatin-remodeling complex. Cell, 1996. 87(7): p. 1249-60.
6. Cairns, B.R., et al., Two actin-related proteins are shared functional components of the chromatin-remodeling complexes RSC and SWI/SNF. Mol Cell, 1998. 2(5): p. 639-51.
7. Cairns, B.R., et al., Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains. Mol Cell, 1999. 4(5): p. 715-23.
8. Chai, B., et al., Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair. Genes Dev, 2005. 19(14): p. 1656-61.
9. Lorch, Y., B. Davis, and R.D. Kornberg, Chromatin remodeling by DNA bending, not twisting. Proc Natl Acad Sci U S A, 2005. 102(5): p. 1329-32.
10. Asturias, F.J., et al., Electron microscopic analysis of the RSC chromatin remodeling complex. Methods Enzymol, 2004. 376: p. 48-62.
11. Strick, T. and A. Quessada-Vial, Chromatin remodeling: RSC Motors along the DNA. Curr Biol, 2006. 16(8): p. R287-9.
12. Angus-Hill, M.L., et al., A Rsc3/Rsc30 zinc cluster dimer reveals novel roles for the chromatin remodeler RSC in gene expression and cell cycle control. Mol Cell, 2001. 7(4): p. 741-51.
13. Moreira, J.M. and S. Holmberg, Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC. Embo J, 1999. 18(10): p. 2836-44.
14. Kasten, M., et al., Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 Lys14. Embo J, 2004. 23(6): p. 1348-59.
15. Soutourina, J., et al., Rsc4 Connects the Chromatin Remodeler RSC to RNA Polymerases. Mol Cell Biol, 2006. 26(13): p. 4920-33.
16. Damelin, M., et al., The genome-wide localization of Rsc9, a component of the RSC chromatin-remodeling complex, changes in response to stress. Mol Cell, 2002. 9(3): p. 563-73.
17. Ng, H.H., et al., Genome-wide location and regulated recruitment of the RSC nucleosome-remodeling complex. Genes Dev, 2002. 16(7): p. 806-19.
18. Hsu, J.M., et al., The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation. Mol Cell Biol, 2003. 23(9): p. 3202-15.
19. Baetz, K.K., et al., The ctf13-30/CTF13 genomic haploinsufficiency modifier screen identifies the yeast chromatin remodeling complex RSC, which is required for the establishment of sister chromatid cohesion. Mol Cell Biol, 2004. 24(3): p. 1232-44.
20. Koyama, H., et al., RSC Nucleosome-remodeling complex plays prominent roles in transcriptional regulation throughout budding yeast gametogenesis. Biosci Biotechnol Biochem, 2004. 68(4): p. 909-19.
21. Lu, Y.M., et al., Dissecting the pet18 mutation in Saccharomyces cerevisiae: HTL1 encodes a 7-kDa polypeptide that interacts with components of the RSC complex. Mol Genet Genomics, 2003. 269(3): p. 321-30.
22. Romeo, M.J., et al., HTL1 encodes a novel factor that interacts with the RSC chromatin remodeling complex in Saccharomyces cerevisiae. Mol Cell Biol, 2002. 22(23): p. 8165-74.
23. Da, G., et al., Structure and function of the SWIRM domain, a conserved protein module found in chromatin regulatory complexes. Proc Natl Acad Sci U S A, 2006. 103(7): p. 2057-62.
24. Boyer, L.A., et al., Essential role for the SANT domain in the functioning of multiple chromatin remodeling enzymes. Mol Cell, 2002. 10(4): p. 935-42.
25. Treich, I. and M. Carlson, Interaction of a Swi3 homolog with Sth1 provides evidence for a Swi/Snf-related complex with an essential function in Saccharomyces cerevisiae. Mol Cell Biol, 1997. 17(4): p. 1768-75.
26. Lin, C.-C., Analysis of Yeast RSC8 mutations affecting Htl1p binding. National Yang-Ming University, School of Life Science, Institute of Genetics, 2005.
27. Taneda, T. and A. Kikuchi, Genetic analysis of RSC58, which encodes a component of a yeast chromatin remodeling complex, and interacts with the transcription factor Swi6. Mol Genet Genomics, 2004. 271(4): p. 479-89.
28. Wilson, B., et al., The RSC chromatin remodeling complex bears an essential fungal-specific protein module with broad functional roles. Genetics, 2006. 172(2): p. 795-809.
29. Tsai, A., Study the relationship between Yeast Htl1p and RSC complex. National Yang-Ming University, School of Life Science, Institute of Genetics, 2002.
30. Puig, O., et al., The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods, 2001. 24(3): p. 218-29.
31. Krogan, N.J., et al., RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol Cell Biol, 2002. 22(20): p. 6979-92.
32. Krogan, N.J., et al., Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature, 2006. 440(7084): p. 637-43.
33. Saha, A., J. Wittmeyer, and B.R. Cairns, Chromatin remodeling by RSC involves ATP-dependent DNA translocation. Genes Dev, 2002. 16(16): p. 2120-34.
34. Shim, E.Y., et al., The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks. Mol Cell Biol, 2005. 25(10): p. 3934-44.
35. Fortes, P., et al., Luc7p, a novel yeast U1 snRNP protein with a role in 5' splice site recognition. Genes Dev, 1999. 13(18): p. 2425-38.