臺灣博碩士論文加值系統

MMS21又稱為Nse2 (non-SMC element 2)，在過去的研究發現是SMC5/6的一個subunit，並且MMS21以其N-terminal region和SMC5結合，失去和SMC5結合能力的MMS21會造成酵母菌的生長缺陷，並且容易有DNA 受損的情形。hMMS21同時也是一個SUMO E3 ligase，缺少hMMS21的細胞DNA容易受到致變因子(例如MMS)的影響而受損，在缺少hMMS21的細胞中送回wild-type hMMS21則能夠救援DNA 容易受損的情形，而送入ligase活性缺失的mutant則無法達到救援的功效，顯示ligase活性在DNA repair中扮演重要角色。
過去實驗室針對hMMS21的研究發現，其對細胞生長會有影響並且參與細胞週期G1-S transition 的調控，缺少hMMS21不僅細胞生長的速率較為緩慢，G1-S transition的時間也有延遲的現象，並且同時影響p21、E2F1等分子的表現。本研究進一步針對hMMS21當中的function domains對細胞週期的影響進行功能性的探討，從細胞生長的分析我們發現，只有full length hMMS21能夠維持細胞的正常生長速率，進一步觀察hMMS21和p21 promoter之間的關係也發現，full length、NSL-deleted 和C215A hMMS21對p21 promoter有抑制作用，而N-terminal domain和C-terminal domain則不會造成顯著的影響，另外，在siRNA knockdown情況下完全缺少hMMS21和表現C-terminal domain的細胞，p21表現量較高，而有表現hMMS21的細胞，p21表現量則有降低情形。
根據本篇研究結果，我們推測維持細胞生長速率單靠C-terminal domain是不夠的，並且也需要細胞核內hMMS21的作用以及SUMO ligase活性。同時也發現，hMMS21可能透過間接的方式調控p21的轉錄，且不需要ligase活性。

MMS21, also named Nse2 (non-SMC element2), is a subunit of SMC5/6 complex and its N-terminal region binds to SMC5. Mutations that affect the interaction between MMS21 and SMC5 lead to growth defects and DNA damage sensitivity. hMMS21 is also a SUMO E3 ligase. In addition, hMMS21 knockdown by siRNA cause DNA damage sensitive in cells. Ectopic expression of wild-type hMMS21 in cells can rescue the hypersensitivity to DNA damage, but not its ligase-inactive mutant. Therefore, the ligase activity of hMMS21 plays an important role in DNA repair.
Studies in our lab have found that hMMS21 can affect cell growth and is involved in the regulation of cell cycle G1-S transition. The cell growth rate is slower and G1-S phase transition is delayed in hMMS21- knockdown cells. Furthermore, the expression level of p21 and E2F1 is also been affected. In this study, we further investigate the role of hMMS21 function domains in cell cycle. Cell growth analysis demonstrates that only full length hMMS21 can keep the normal growth rate of cells. Moreover, in the study of connection between hMMS21 and p21 promoter, we find that full length, NSL-deleted and C215A hMMS21 can inhibit p21 promoter, but not N-terminal domain and C-terminal domain. In hMMS21-knockdown cells, we also demonstrates that p21 level is higher. Ectopic expression of full length hMMS21 can lower p21 level, but not its C-terminal domain.
Our finding suggests that nuclear hMMS21 and ligase activity is essential for keeping normal cell growth rate. Moreover, hMMS21 may regulate p21 promoter indirectly and its ligase activity is dispensible for the regulation of p21 promoter.

目錄 ------------------------------------------- 1
中文摘要 --------------------------------------- 2
英文摘要 --------------------------------------- 3

壹. 緒論 ------------------------------------- 4
貳. 研究動機----------------------------------- 9
參. 實驗材料與方法 --------------------------- 10
肆. 實驗結果 --------------------------------- 20
伍. 討論 ------------------------------------- 25
陸. 參考文獻 --------------------------------- 29
柒. 附圖 ------------------------------------- 33

Andrews, E.A., Palecek, J., Sergeant, J., Taylor, E., Lehmann, A.R., and Watts, F.Z. (2004). Nse2, a Component of the Smc5-6 Complex, Is a SUMO Ligase Required for the Response to DNA Damage. Molecular and Cellular Biology 25, 185-196.

Bohren, K.M. (2004). A M55V Polymorphism in a Novel SUMO Gene (SUMO-4) Differentially Activates Heat Shock Transcription Factors and Is Associated with Susceptibility to Type I Diabetes Mellitus. Journal of Biological Chemistry 279, 27233-27238.

Cazzalini, O., Scovassi, A.I., Savio, M., Stivala, L.A., and Prosperi, E. (2010). Multiple roles of the cell cycle inhibitor p21CDKN1A in the DNA damage response. Mutation Research/Reviews in Mutation Research 704, 12-20.

Dou, H., Huang, C., Van Nguyen, T., Lu, L.-S., and Yeh, E.T.H. (2011). SUMOylation and de-SUMOylation in response to DNA damage. FEBS Letters 585, 2891-2896.

Duan, X., Sarangi, P., Liu, X., Rangi, G.K., Zhao, X., and Ye, H. (2009). Structural and Functional Insights into the Roles of the Mms21 Subunit of the Smc5/6 Complex. Molecular Cell 35, 657-668.

Gill, G. (2004). SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes & Development 18, 2046-2059.


Hirano, T. (2002). The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair. Genes & Development 16, 399-414.

Hochstrasser, M. (2001). SP-RING for SUMO:New Functions Bloom for a Ubiquitin-like Protein. Cell 107, 5-8.

Jürgen Dohmen, R. (2004). SUMO protein modification. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1695, 113-131.

Johnson, E.S. (2004). Protein modification by sumo. Annual Review of Biochemistry 73, 355-382.

Johnson, E.S., and A. A. Gupta. (2001). An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106, 735-744.

Katrien Vermeulen, D.R.V.B.a.Z.N.B. (2003). The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 36, 131-149.

Kaufmann WK, P.R. (1996). DNA damage and cell cycle checkpoints. FASEB J 10, 238-247.

MANUEL ARELLANO, S.M. (1997). Regulation of CDK/cyclin Complexes During the Cell Cycle. Int J Biochem Cdl Bid 29, 559-573.

MASAAKI MATSUOKA, J.-Y.K., ROBERT P. FISHER,DAVID 0. MORGAN, AND CHARLES J. SHERRl (1994). Activation of Cyclin-Dependent Kinase 4 (cdk4) by Mouse M015-Associated Kinase. MOLECULAR AND CELLULAR BIOLOGY 14, 7265-7275.

Piccoli, G.D., Torres-Rosell, J., and Aragón, L. (2009). The unnamed complex: what do we know about Smc5-Smc6? Chromosome Research 17, 251-263.

Pichler, A., A. Gast, J. S. Seeler, A. Dejean, and F. Melchior. (2002). The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109-120.

Potts, P.R., and Yu, H. (2005). Human MMS21/NSE2 Is a SUMO Ligase Required for DNA Repair. Molecular and Cellular Biology 25, 7021-7032.

Roberts, C.J.S.a.J.M. (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13, 1501-1512.

Saitoh, H., D. B. Sparrow, T. Shiomi, R. T. Pu, T. Nishimoto, T. J. Mohun,and M. Dasso. (1998). Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2. Curr Biol 8, 121-124.

Schmidt, D. (2002). Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proceedings of the National Academy of Sciences 99, 2872-2877.

Sergeant, J., Taylor, E., Palecek, J., Fousteri, M., Andrews, E.A., Sweeney, S., Shinagawa, H., Watts, F.Z., and Lehmann, A.R. (2004). Composition and Architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) Complex. Molecular and Cellular Biology 25, 172-184.

Stephan, A.K., Kliszczak, M., and Morrison, C.G. (2011). The Nse2/Mms21 SUMO ligase of the Smc5/6 complex in the maintenance of genome stability. FEBS Letters 585, 2907-2913.

Takahashi, Y. (2003). Comparative Analysis of Yeast PIAS-Type SUMO Ligases In Vivo and In Vitro. Journal of Biochemistry 133, 415-422.