| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
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.
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