Abstract
瞭解哺乳類細胞對輻射線所產生傷害的修補機制是很重要的.為了達到這 個目的,科學家不斷的去尋找對輻射線敏感的突變株,當這些對輻射線敏感 的突變株被陸續分離出來以後,科學家便利用這些突變株與其野生株之間 的相異之處而找出一些可能在修補機制上扮演主要角色的分子.野生型中 國倉鼠卵巢細胞(CHO-K1)及其對輻射線敏感的突變株(xrs -6)在本篇論文 中為實驗材料之一.目前已知這些xrs mutant是double strand break修補 有缺陷,並且除了對輻射線敏感以外同時也對UV.MMS EMS有cross- sensitive的現象.另外有11個mRNA也在xrs-6中表現較CHO- K1來的低.在 本研究中我們分離出xrs-6的輻射敏感度自然返回株去進一步探討xrs mutant的特性.我們實驗的結果顯示,自然返回株的dsb修補的能力與CHO- K1是相同的.可是這11個mRNA並沒有回到與CHO-K1一樣的含量.代表這11個 mRNA與細胞的輻射敏感度較無關係.我們發現自然返回株與CHO-K1一樣具 有DNA end binding activity代表其抗輻射的機制應與CHO-K1是相同的, 現在已知DEB activity主要是由Ku protein所造成的,而xrs-6的缺陷主要 在Ku80這個subunit上,自然返回株除了對輻射線是具有抗性外對紫外線也 是具有抗性但對MMS EMS仍然是敏感的.這結果似乎暗示輻射線與紫外線的 修補路徑有共通點而MMS及EMS的修補路徑則不經過這共通點,而Ku protein的功能似乎與此共通點有關. Understanding of the molecular mechanism of radiation sen sitivity in mammalian cells is important both in clinical and basic research.The wild type Chinese hamster ovary(CHO)K1 cell line and its x-ray sensitive mutant xrs-6 were used as the model for this purpose.In addition to x-ray, this mutant is cross-sensitive to other DNA-damaging agents, such as UV,MMS and EMS.It has been shown that xrs-6 mutant was defective in rejoining DNA double strand break(dsb)and in expression of 11 genes.Recently,the defect in repairing DNA dsb was found due to lack of the DNA-end binding(DEB) activity resulted from the deficiency of Ku80 protein.In this work, a spontaneous revertant SR-16 from xrs-6 was isolated in order to futher characterize the mutation. The SR-16 revertant was found to have the same sensitivity to x-ray and UV as CHO-K1 cells, but was still sensitive to MMS and EMS. Furthermore, SR-16 cells had the same DEB activity and DNA dsb repair capacity as the CHO-K1 cells. The results suggest that the mechanism of radio- resistance in SR-16 is the same as in CHO-K1 cells and further confirm the deficiency of Ku is responsible for the x- raynsitive phenotype of the xrs-6 mutant. The level of 11 mRNAs in SR-16 cells did not go back to the same level as in the CHO- K1 cell line. The expression of those 11 mRNAs is there- fore not regulated by Ku, although Ku protein has been shown to be able to regulate transcription. The results also suggest that there may be a common point between the repair of damages caused by x-ray and UV, and Ku is involved in both repair pathway.