Molecular and cellular analysis of Cockayne syndrome

由于DNA修复的未知缺陷，Cockayne综合征(CS)细胞对紫外线(UV)辐射表现出超敏反应。我们发现CS细胞对腺苷(AC)或胸腺嘧啶核苷(DT)也很敏感。CS细胞的紫外线抗性突变株对AC或DT具有抗性，表明CS细胞的AC或DT超敏反应与其DNA修复缺陷直接相关。由于AC是DNA聚合酶的特异性抑制物，或作用于核糖核苷酸还原酶(RNR)作为dNTP类似物，因此我们检测了CS细胞的RNR活性和dNTP池。CS细胞的POL&lt；α-GT；、RNR活性和dNTP池水平略低于正常细胞。UV照射后这些参数的时程测量表明，照射后5-12小时，CS细胞的RNR活性没有恢复，而Poll&lt；α&gt；活性则恢复正常。正常细胞和CS细胞紫外线照射后dNTP池的初始下降和随后的增加没有显著差异。这些结果表明，CS细胞可能存在RNR异常，如RNR M1亚基变构结构域异常或RNR基因阻滞子异常。由于自然产生的AC抗性CS克隆的出现频率较高，将正常细胞DNA导入CS细胞以分离AC抗性CS细胞和挽救CS基因的方法失败。最近获得了小鼠RNR M1和M2亚基的cDNA。我们将检测CS细胞对AC、DT或UV的敏感性是否可以通过这些cDNA的导入而恢复，并比较CS细胞和正常细胞在紫外线照射后不同时间的RNR基因组及其转录。

Cockayne syndrome(CS)cells show hypersensitivity to ultraviolet-light(UV)irradiation due to the unknown defect in DNA repair. We found that CS cells are also hypersensitive to aphidicolin(AC)or thymidine(dT). UV-resistant revertant of CS cells was shown to be resistant to AC or dT, indicating that AC- or dT-hypersensitivity of CS cells is directly correlated with their DNA repair defect. Since AC is a specific inhibitor of DNA polymerase <alpha> (pol <alpha> ) or act on ribonucleotide reductase(RNR) as a dNTP analogue, pol <alpha> , RNR activities and dNTP pool in CS cells were examined. CS cells showed slightly lower levels of pol <alpha> , RNR activities and dNTP pool than normal cells. The time course measurements of these parameters after UV-irradiation revealed that there was no recovery of RNR activity in UV-irradiated CS cells, while the pol <alpha> activity was recovered normally in UV-irradiated CS cells 5 - 12 hrs after UV-irradiation. No significant differences in the initial decrease and subsequent increase of dNTP pool after UV-irradiation were observed between normal and CS cells. All these data indicate that CS cells may have abnormal RNR, for example, abnormal allosteric domain of M1 subunit of RNR, or abnormal repressor for RNR gene.Transfection of normal cellular DNA into CS cells to isolate the AC-resistant CS cells and to rescue the CS gene were failed because of the high frequency appearance of spontaneous AC-resistant CS clones. The cDNAs for mouse M1 and M2 subunits of RNR were recently obtained. We are going to examine whether AC, dT or UV-sensitivities of CS cells may be recovered by the introduction of these cDNAs and also to compare the RNR genome and its transcription at various time after UV-irradiation between CS and normal cells.