Affinity Purification of DNA Repair Proteins from Chlorella pyrenoidosa and Their Functrional Analysis

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Project title

Code/計畫編號

NSC89-2311-B019-004

Translated Name/計畫中文名

小球單胞藻DNA修補蛋白之親和性純化與功能鑑定

Project Coordinator/計畫主持人

Todd Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=580409

Year

2000

Start date/計畫起

01-08-2000

Expected Completion/計畫迄

01-07-2001

Bugetid/研究經費

741千元

ResearchField/研究領域

生物科學

先前研究已完成以化學藥物 cisplatin 處理過之質體 DNA 為修補基質，建立在小球單胞藻蛋白抽取液中檢測核酸切割修補作用之體外 DNA 修補試驗，我們將 UV 照射過之 DNA 固定在小顆粒上，再以親和性吸附法吸取小球藻抽取液中之 DNA 修補蛋白; 未吸附之抽取液與經正常 DNA 吸附後之抽取液都有極明顯之修補作用，抽取液在吸附後幾乎無法修補受 cisplatin 處理之質體，強度約為吸附前之 2%。以蛋白質電泳分析附著在正常與 UV 照射過 DNA 上之蛋白，發現有一 72 kDa 蛋白質對受傷 DNA 有特別強之親和力，因為此蛋白與修補活性下袶之關聯，此一 72 kDa 之蛋白應參與小球藻之核酸切割修補作用(研究成果已發表於 Plant Science 156:95-102,2000)。由於 UV 照射 DNA 主要產生 cyclobutane pyrimidine dimer 與 6-4 photoproduct 兩種傷害，本研究繼續以帶有 CPD 或 6-4PP 之 DNA 藉親和性吸附與雙向蛋白質電泳分離法探討小球單胞藻中是否存在不同核酸切割修補群，結果發現吸附在 CPD 與 6-4PP 上之蛋白質分布型式確有差異，而此蛋白質分布差異經雙向蛋白質電泳法在等電點 3-10 間分離後已確認，目前正以委託方式利用質譜儀撞擊資料分析蛋白質可能之功能。另外在本計劃經費補助下，我們也對斑馬魚(Danio rerio) 中之 UV 辨識蛋白進行研究，結果偵測到一種胚胎專一性辨識蛋白群，對 CPD 與 6-4PP 有類似之結合能力，但在成熟後斑馬魚中此辨識蛋白群之表現明顯下降，斑馬魚反而表現出與真核生物類似之 DNA 修補系統，故此胚胎專一性辨識蛋白群可能參予胚胎 DNA 修補作用 (研究成果已投稿 Fish Physiol. Biochem. Manuscript No. FISH 140-01 修改中)。capacity, yet the extracts post incubation with UV-damaged DNA almost failed to repair the damaged DNA. A polypeptide estimated to be 72 kDa in molecular mass was found to bind much more strongly to the damaged DNA than to the unirradiated DNA after analyzing the proteins bound to the solid matrix by SDS-PAGE, and this polypeptide is believed to be important to NER in C. pyrenoidosa. Because cyclobutane pyrimidine dimmer (CPD) and 6-4 photoproduct (6-4PP) are two major forms of DNA damage generated after UV irradiation, this research continued to examine if different NER systems existed in C. pyrenoidosa by affinity adsorption using CPD and 6-4PP-specific DNA ligand. Differential polypeptide patterns were observed after affinity adsorption with DNA probes carrying different types of lesion and the presence of some lesion-specific proteins were confirmed by two-dimensional gel electrophoresis. The functions of these proteins are being studied by protein mass spectrometry. Under the financial support of this grant, a UV-damaged-DNA binding activity was detected in the embryonic extracts of zebrafish(Danio rerio) and this binding activity was thought to exist as a protein complex that showed a similar affinity for both CPD and 6-4PP. A NER system conserved among eukaryotic organisms was found to predominate in zebrafish beyond the embryonic stage.

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Affinity Purification of DNA Repair Proteins from Chlorella pyrenoidosa and Their Functrional Analysis

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