Abstract
DNA 依存性蛋白激酵素 (DNA-dependent protein kinase; DNA-PK)是一絲氨酸/蘇氨酸蛋白激酵素 (serine/threonine protein kinase)係由其催化次單元 DNA-PKcs (460 kDa) 及一個DNA結合蛋白 Ku (p70/p86)所組成。 從試管中的實驗指出 DNA 依存性蛋白激酵素可磷酸化一個或多個與 DNA 雙股斷裂修補 (double-stranded break repair; DSBR) 及 V(D)J 重組中需要的蛋白質。根據這些試管中的研究，DNA 依存性蛋白激酵素可能擔任調節 DNA rejoining 及以本身當成骨架將其他 DNA 修補/重組蛋白質維持在DNA雙股斷裂的位置以利進行DNA的修補。DNA 依存性蛋白激酵素的體內受質到目前為止還不為人所知。 首先，我們的策略是利用雙向電泳的方式來分離並且比較抗輻射細胞株Ku804與輻射敏感細胞株xrs-6（Ku86缺失）的32P磷酸標誌核蛋白。如果某個磷酸核蛋白在 Ku804中出現卻在 xrs-6 中找不到，這個磷酸核蛋白就有可能是DNA依存性蛋白激酵素的體內受質。第二，我們利用 SDS-PAGE 比較 DNA 依存性蛋白激酵素正常及缺失細胞株中與雙股 DNA 結合的磷酸蛋白。第三，為了探索DNA 依存性蛋白酵素當成骨架來維持 DNA 修補蛋白的可能性，我們利用anti-Ku 的抗體來進行共免疫沈澱法 (co-immunoprecipitation) 來研究。最後，以共純化 (co-purification) DNA 依存性蛋白激酵素的方法來尋找與其有相互作用的蛋白質。藉著以上數種實驗的方法，我們已經找到兩個我們有興趣的蛋白質，其分子量為 100 及 46 kDa。DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a catalytic subunit DNA-PKcs (p460) and a DNA binding component Ku (p70/p86). In vitroexperiments have indicated that DNA-PK phosphorylates one ormore components required for DNA double-stranded break repair(DSBR) and V(D)J recombination. Based on these and other invitro studies, it has been proposed that DNA-PK might serve as amodulator of DNA rejoining and a scaffold that recruits theother repair/recombination proteins to the DNA DSB sites. The invivo substrates of this kinase is remain unknown. In thisstudy, Ku80-defective mutant CHO xrs-6 (X-ray sensitive) and itsX-ray resistant transfectant xrs-6/Ku80 were compared in nuclearphosphoproteins and double-stranded DNA (ds-DNA) bindingphosphoproteins, using 2D electrophoresis and SDS-PAGEtechniques, respectively. Third, to explore the possibility thatDNA-PK serves as a scaffold to recruit repair proteins,coimmunoprecipitation of ds-DNA binding proteins with anti-Kuantibody was used. Finally, co-purification of protein with DNA-PK was used in order to find proteins interacting with DNA-PK.Using these approaches, we have identified two proteins ofinterest. The molecular weight of these two proteins are 100 and46 kDa.