PIR51在DNA修補途徑的功能探討

中國醫藥大學機構典藏 China Medical University Repository, Taiwan > 生命科學院 > 生物科技學系暨碩士班 > 研究計畫 > Item 310903500/11277

jsp.display-item.identifier=請使用永久網址來引用或連結此文件: http://ir.cmu.edu.tw/ir/handle/310903500/11277

题名:	PIR51在DNA修補途徑的功能探討
作者:	蔡士彰
贡献者:	生命科學院生物科技學系
关键词:	DNA 破壞和修復;同源重組;誘導細胞株;免疫沉澱法;蛋白質的穩定性;DNA damage and repair;homologous recombination;inducible cell line,immunoprecipitation,protein stability
日期:	2009-07-31
上传时间:	2009-09-01 15:09:21 (UTC+8)
摘要:	Pir51 是一個存在Rad51 複合體，但功能未知蛋白質。我們篩選從侵襲性與和緩性淋巴瘤病人樣品後，發現Pir51 高度表達在侵襲性淋巴瘤病人。結果顯示，Pir51 和Rad51 表示幾乎相同地被調控在細胞週期期間。我使用siRNA 去除細胞內Pir51 顯示，Pir51 增加mitomycin C 敏感性，進而得知Pir51 牽涉DNA 交鍵修復。Rad51 與腫瘤遏抑基因BRCA2 交互作用。BRCA2 在淋巴瘤、和攝護腺, 胰臟, 乳房, 和卵巢癌扮演重要角色。初步結果顯示， Pir51 與BRCA2 的蛋白質相互作用。我的提案著重於探討Pir51 在DNA 破壞和修復的生物性功能，並提供新洞察方向。第一年(2007年8月～2008年7月)目標: 做Pir51 剔除細胞株和Pir51 誘導表達細胞株我將使用二種方法探討Pir51 蛋白質的功能。一種是功能喪失策略,另一種是功能獲取策略。在功能喪失策略, 我們將做可誘導Pir51 剔除細胞株受四圜素調控。siPir51 表達質體已被構建和測試調控Pir51 蛋白質表達。在沒有四圜素時,遏抑因子緊緊與四圜素操作因子序列結合進而抑制基因表達。另一方法是功能獲取策略，將構建誘導Pir51 蛋白質表達的細胞株。第二年(2008年8月～2009 年7月)目標: 鑑定與Pir51 結合蛋白質 Rad51 已被證明與很多蛋白質結合包括RPA 、p53 、BRCA2 、ATM 、BRCA1 、和Ubc9。因為 Pir51 與Rad51 結合, 我們將使用免疫沉澱法去辨認Pir51 複合體。另外,我們將測試蛋白質譬如 BRCA1 、BRCA2 、FancD2, 和DSS 與Pir51 的結合情形，進而瞭解Pir51 蛋白質在DNA 修復途徑的生物性角色。我們將獲得很多資訊從Pir51 結合的蛋白質並將我們探索Pir51 的不同生物角色。第三年(2009年8月～2010 年7月)目標: 描繪Pir51 蛋白質的穩定性和確定Pir51 缺陷在DNA 受損的反應我們將由二個方向檢視Pir51 蛋白質的穩定性: ubiqitination 和phosphorylation 。另外, 我們將由使用Pir51 惕除細胞鑑認Pir51 在同源重組的特定階段和在非同源末端重組的作用。這些研究提供對在DNA 受損和修復控制的複雜機制之詳盡的理解。 Pir51 is a protein of previously unknown function that exists in a complex with Rad51. Recently, we identified Pir51 using a screen for genes that are highly expressed in aggressive lymphoma compared to indolent lymphoma patient samples. We showed that Pir51 and Rad51 expression are almost identically regulated during the cell cycle. I used siRNA depletion to show that Pir51 regulates mitomycin C sensitivity and DNA crosslink repair. In addition, Rad51 binds BRCA2, a tumor suppressor playing an important role in lymphoma, prostate, pancreatic, breast, and ovarian cancers. Based on the preliminary data, I find that Pir51 interacts with BRCA2. I propose to characterize the biological functions of Pir51 in DNA repair pathways. My proposal will provide essential new insights for the Pir51 protein in the control of DNA damage and repair. First year (2007年8月～2008年7月) SPECIFIC AIM 1: Make a Pir51 knockout inducible RNAi cell line and an inducible expression cell line. I will use two approaches to characterize functions of the Pir51 protein. One is the loss of function strategy, and the other is the gain of function strategy. In the loss of function method, we will make inducible Pir51 knockout cells under the tightly regulated, tetracycline-controlled gene expression system. A siPir51 expression plasmid has been constructed and tested for down-regulating the protein level of Pir51. In the absence of tetracycline, the tetracycline-controlled transcriptional suppressor tightly binds tetracycline operator sequences and locks gene transcription. The other approach is using the gain of function strategy to make a constitutive or/ and an inducible cell line expressing the FLAG-tagged Pir51 protein. Second year (2008 年8 月～2009 年7 月) SPECIFIC AIM 2: Identification of proteins that interact with Pir51. Rad51 has been reported to biochemically interact with a large number of proteins including RPA, p53, BRCA2, ATM, BRCA1, and Ubc9. Since Pir51 binds to Rad51, we will immunoprecipitate and identify the Pir51 complex. In addition, while we immunoprecipitated the Pir51 complex, we will examine the proteins such as BRCA1, BRCA2, FancD2, and DSS to characterize the roles of Pir51 in DNA repair. We will obtain a lot of information from Pir51 interacting proteins and explore different roles of Pir51. Third year (2009年8月～2010 年7 月) SPECIFIC AIM 3: Characterize the protein stability of Pir51 and determine the defects of Pir51 in the DNA damage response. The protein stability plays a crucial role in biological functions.We will examine the protein stability of Pir51 by two directions: ubiqitination and phosphorylation. In addition, we will characterize the functions of Pir51 in the specific step of homologous recombination and in non-homologous end joining by using Pir51 null cells. These studies will ultimately provide essential new insight for a thorough understanding of the intricate mechanisms in the control of DNA damage and repair.
显示于类别:	[生物科技學系暨碩士班] 研究計畫

文件中的档案:

档案	描述	大小	格式	浏览次数
index.html		0Kb	HTML	181	检视/开启

在CMUR中所有的数据项都受到原著作权保护.

TAIR相关文章

数据加载中.....