Characterization of Early-Responsive Proteins Induced in C. pyrenoidosa by Low Doses of UV-C

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Project title

Code/計畫編號

NSC91-2311-B019-001

Translated Name/計畫中文名

低量紫外線C早期引誘發小球藻蛋白特性鑑定

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=768459

Year

2002

Start date/計畫起

01-08-2002

Expected Completion/計畫迄

01-07-2003

Bugetid/研究經費

941千元

ResearchField/研究領域

生物科學

由於UV照射DNA主要產生cyclobutane pyrimidine dimer與6-4 photoproduct兩種傷害，本研究以帶有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蛋白群，結果發現吸附在CPD 與6-4PP上之蛋白質分布型式確有差異，且實驗顯示一群分子量約62 kDa與一50 kDa之蛋白似對6-4PP有較佳專一性，而吸附後蛋白質分布差異經雙向蛋白質電泳法在等電點4,7-5.9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這些蛋白質可能之功能而其中之一50 kDa蛋白與綠色植物之ATP synthase有明顯氨基酸序列相似性。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，成熟後斑馬魚反而表現出與真核生物類似之DNA修補蛋白XP-A，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.並接受發表)。 Cyclobutane pyrimidine dimmer (CPD) and 6-4 photoproduct (6-4PP) are two major forms of DNA damage generated after UV irradiation, this research attempted to examine if different NER systems existed in unicellular alga C. pyrenoidosa by affinity adsorption and 2-D gel electrophoresis using CPD and 6-4PP-specific DNA ligands Differential polypeptide patterns were observed after affinity adsorption with DNA probes carrying different types of lesion and one 70 kDa polypeptide seemed to bind more strongly to 6-4PP than to CPD as shown by affinity adsorption and SDS-PAGE. The presence of some lesion-specific proteins with pI between pH 5.2 and 5.4 were confirmed by two-dimensional gel electrophoresis. The possible functions of these 6-4PP-binding proteins are being studied by protein mass spectrometry and one 50-kDa polypeptide was shown by Mascot search to have a significant sequence homology with ATP synthases in green plants. Under the financial support of this grant, a UV-damaged-DNA binding activity highly expressed in zebrafish(Danio rerio) early embryos was detected by gel shift assay and this binding activity showed a similar affinity for both CPD and 6-4PP. The embryonic UV- binding activity was found to exist as two protein complexes having overlapping components as shown by a UV crosslinking experiment. The expression of NER DNA damage-recognition protein XP-A conserved among eukaryotic organisms was found to arise only in zebrafish beyond the embryonic stage. Hence, the embryonic UV-damaged-DNA binding activity might be responsible for DNA repair in zebrafish embryos.

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Characterization of Early-Responsive Proteins Induced in C. pyrenoidosa by Low Doses of UV-C

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