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標題: 十字花科黑腐病菌SOS調控蛋白LexA的純化與結晶
Purification and crystallization of SOS response regulators LexA repressors from Xanthomonas campestris
作者: 王久銘
wang, chio-ming
關鍵字: Xanthomonas campestris;十字花科黑腐病菌;SOS response regulators;LexA repressors;SOS調控蛋白;LexA
出版社: 生物化學研究所
引用: 1.Little, J. W. & Mount, D. W. (1982). The SOS reguiatory system of Escherichia coli. Cell 29, 11-22. 2.Mohana-Borges, R., Pacheco, A. B. F., Sousa, F. J. R., Foguel, D., Almeida, D. F. & Silva, J. L. (2000). LexA Repressor Forms Stable Dimers in Solution. J.Biol.Chem. 275, 4708-4712. 3.Little, J. W. (1991). Mechanism of specific LexA Cleavage: autodigestion and the role of RecA coprotease. Biochimie 73, 411-422. 4.Little, J. W. (1993). LexA Cleavage and Other Self-Processing Reactions. JOURNAL OF BACTERIOLOGY 175, 4943-4950. 5.Rehrauer, W. M., Lavery, P. E., Palmer, E. L., Singh, R. N. & Kowalczykowski, S. C. (1996). Interaction of Escherichia coli RecA protein with LexA Repressor. J.Biol.Chem. 271, 23865-23873. 6.Peat, T. S., Frank, E. G., McDonald, J. P., Levine, A. S., Woodgate, R. & Hendrickson, W. A. (1996). Structure of the UmuD'' protein and its regulation in response to DNA damage. Nature 380, 727-730. 7.LOU, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W. & Strynadka, N. C. J. (2001). Crystal Structure of LexA: A Conformational Switch for Regulation of Self-Cleavage. Cell 106, 585-594. 8.Shepley, D. P. & Little, J. W. (1996). Mutant LexA proteins with specific defects in autodigestion. PNAS 93, 11528-11533. 9.Slilaty, S. N. & Little, J. W. (1987). Lysine-156 and serine119 are required for LexA Repressor Cleavage: A possible mechanism. PNAS 84, 3987-3991. 10.Little, J. W. (1984). Autodigestion of lexA and phage lamda repressors. PNAS 81, 1375-1379. 11.Kim, B. & Little, J. W. (1993). LexA and lambda CI Repressor as Enzymes: Specific Cleavage in an intermolecular Reaction. Cell 73, 1165-1173. 12.Brent, R. & Ptashne, M. (1981). Mechanism of action of the lexA gene product. PNAS 78, 4204-4208. 13.Fogh, R. H., Ottleben, G., Ruterjansl, H., Schnarr, M., Boelens, R. & Kaptein, R. (1994). Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy. EMBO 13, 3936-3944. 14廖怡雯 (2005).十字花科黑腐病菌緊急求救反應之探討。國立中興大學分子生物研究所碩士論文 15王雲慶 (2006) Xanthomonas campestris pv. Campestris中受到lexA1與lexA2調控之基因之探討。國立中興大學分子生物研究所碩士論文
當細菌在逆境中DNA造成損害時,會啟動因應的修補機制。其中SOS response為可被誘導的RecA-LexA dependent的DNA修補機制。E. coli的LexA平常以dimer形式,結合在SOS系統調控區域的SOS box上,抑制20個以上SOS修補基因包括lexA、recA、uvrA、sfiA的轉錄。但是當RecA以ATP-dependent的方式,與損毀的單股DNA結合成絲狀時,被活化的RecA會促使LexA水解,因而逐次解除被抑制的SOS response基因,LexA藉此調控不同層次的DNA修補機制。
十字花科黑腐病菌 (Xanthomonas campestris pv. campestris) 帶有兩個LexA家族蛋白-LexA1及LexA2,長度分別為221和209氨基酸,序列與E. coli的LexA近似性為63%,唯仍不清楚xcc菌中LexA1與LexA2在SOS response的關連,以及這兩個蛋白質在結構上的同異性。在本計畫中,已分別大量表達LexA1及LexA2蛋白質,經由樹脂層析純化及濃縮至18 及20 mg/mL,並以蒸氣昇華法篩選結晶條件。經測試Polyethylene Glycol 400-8000、(NH4)2SO4及K2HPO4等沉澱劑,晶珠呈現油性分離或amorphous沉澱。同期純化全長LexA2時,我們另行純化經trypsin切除DNA-binding domain的LexA2,得到濃度為16.4 mg/mL具有peptidase domain的LexA2,經測試不同的結晶條件後,仍得到表徵近似的晶珠。因此,進一步搜尋適當的結晶溶液配方及沉澱劑,能破除結晶臨界的油水分離相位,將是結晶LexA1及LexA2蛋白質的主要關鍵。

When bacteria are exposed to heavy doses of DNA-damaging agents, their damaged DNA induce an array of DNA repair responses. Among these, inducible SOS response is RecA-LexA dependent and error-prone. Normally, LexA binds to the SOS boxes as a dimmer and negatively regulates expression of 20 SOS genes inCluding lexA, recA, uvrA, and sfiA. The filamentous RecA is activated in an ATP-dependent fashion after DNA damage and acts as a coprotease in the autocatalytic digestion of the LexA repressors, which subsequently relieves the repression of the SOS genes and activate different DNA repair mechanisms according to the level of LexA affinity for the SOS boxes.
Xanthomonas campestris pv. campestris str17 (xcc) has two LexA repressors- LexA1 (221 aa) and LexA2 (209 aa). Their sequences are highly homologous to that of the E. coli LexA 63% identity. The interplay of LexA1 and LexA2 in the SOS response and their structural features remain elusive. In this study, I expressed the full-length LexA1 and LexA2 in large quantity, subsequently purified the proteins by affinity and ion-exchange chromatography to obtain 18 mg/mL and 20 mg/mL, respectively. Crystallization trials using vapor sublimation showed common features of amorphous precipitation or phase separation in various precipitants inCluding polyethylene glycol 400-8000, (NH4)2SO4 and K2HPO4 . Initial screening of the purified trypsin-digested LexA2 protein containing the C-terminal peptidase domain (16.4 mg/mL) also showed similar features in the crystallization drops. Thus, to reduce the oily phase separation would be crucial for ordered crystal growth of the Xanthomonas LexA1 and LexA2.
其他識別: U0005-0908200714552200
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