Abstract
環境中有許多物質都會造成DNA的傷害，例如紫外線、X 射線或烷基化物 。而細胞中有特定的修補機制會來修補這些 DNA 傷害。例如紫外線的傷 害是經由核甘酸切除修補機制，而X射線和烷基化物質的傷害則是由鹼基 切除修補機制來進行。我利用 DNA 修補合成分析法與 DNA 缺口連接酵素 分析法來檢查中國倉鼠卵巢細胞(CHO)、對X射線敏感細胞(XRS-5)、紫外 線敏感細胞(UVL-10)，和人類子宮頸癌細胞 (HeLa) 的萃取液中，DNA 修 補酵素的活性是否正常。結果發現，CHO- K1 和 HeLa 細胞萃取液，對紫 外線、X 射線以及烷基化物質的傷害修補活性皆正常。而 XRS-5 細胞萃 取液則對 X 射線和紫外線傷害的修補活性很低。UVL-10細胞萃取液則對 紫外線傷害的DNA修補活性很差。 CHO-K1 細胞萃取液的三種 DNA 接合酵 素活性正常，而 HeLa 細胞 DNA 接合酵素 II + III 活性很低。XRS-5 和UVL-10 細胞萃取液的三種 DNA 接合酵素活性皆比 CHO-K1 的低。進一 步分析 DNA 接合酵素與 AMP 結合能力發現， CHO-K1 細胞萃取液中的 DNA接合酵素皆可和 AMP 結合，但 XRS-5 的DNA 接合酵素 II 與 UVL-10 的 DNA 接合酵素 I 和 II 皆無法和 AMP 結合形成複合物。本實 驗的結果顯示 XRS-5 細胞之所以對 X 射線特別敏感可能與對 X 射線所 誘發的 DNA 修補和成酵素活性低有關。 而 UVL-10 細胞之所以對紫外線 特別敏感可能與對紫外線所誘發的 DNA 修補和成酵素活性低有關。 DNA damages induced by ultraviolet light (UV) are mainly mended by necleotide-excision-repair process, whereas those induced by X-ray and alkylating agents are mainly mended by base-excision- repair process. Using cell extracts, I have attempted to identify the defects of Chinese hamster ovary cells sensitive to UV and X-ray. The results indicate that cell extracts from CHO-K1 and HeLa were proficient in the repair synthesis of plasmid damaged with UV, X-ray, and alkylating agent, methyl methanesulfonate (MMS). However, extracts from XRS-5 cells, an X-ray sensitive CHO cell line, were deficient in the repair synthesis of plasmids damaged with X-ray and UV, and extracts from UVL-10 cells, a UV- sensitive CHO cell line, were deficient in the repair synthesis of UV-damaged plasmids. While extracts from CHO-K1 cells were also proficient in DNA ligation, extracts from HeLa cells were deficient in the activities of DNA ligases II + III. Extracts from XRS-5 and UVL-10 were also with lower activities in DNA ligases II + III. While the DNA ligases I, II and III in the extract from CHO-K1 cells could form complexes with AMP, the complex formation was not detected for the ligase II from in the extracts from XRS-5, and for the ligases I and II in the extracts from UVL-10. While the complex formation of ligases I, II and III in extracts from CHO-K1 and HeLa cells with AMP increased with increasing time for reaction, this was not observed for ligases I and III in extracts from XRS-5. In the extracts from UVL-10, the complex formation of ligase III with AMP even decreased with increasing time for reaction.