Analysis of mechanisms of DNA repair and UV-induced mutation by a cell-free system.

建立了DNA修复、复制和突变的体外无细胞检测体系。构建了以细菌supF基因为突变靶点、以SV40复制起点和T抗原基因为在人细胞中复制的基因的质粒<32>pMY189，将该质粒与正常人细胞的提取物一起培养，当质粒与着色性干皮病（XP）细胞的提取物一起孵育时，没有发生这种情况。当<32>质粒与来自两个不同遗传互补组的XP细胞的提取物的混合物一起孵育时，[^ P] dATP掺入到UV照射的质粒中。这些结果表明，在无细胞提取物中发生的DNA修复类似于活细胞中的DNA修复。我们发现p53蛋白在无细胞系统中对DNA修复机制没有影响，尽管p53蛋白降低了人类细胞中紫外线诱导的突变频率。<32>当质粒与SV 40 T抗原和正常或XP细胞提取物一起孵育时，[^ P] dATP也通过DNA复制掺入未照射的质粒pMY189中。我们发现从S期细胞中获得的提取物在无细胞体系中具有进行DNA复制的高活性。

We have established the in vitro cell-free assay systems for DNA repair, replication and mutation. The plasmid pMY189 which has a bacterial supF gene as a mutation target and SV40 replication origin and T antigen gene for replication in human cells was constructed and used in the system.Incorporation of [^<32>P] dATP to the UV-irradiated pMY189 plasmid by unscheduled DNA synthesis occurred when the plasmid was incubated with the extract from normal human cells, however, it did not occur when the plasmid was incubated with the extract from xeroderma pigmentosum (XP) cells. The incorporation of [^<32>P] dATP to the UV-irradiated plasmid was occurred when the plasmid was incubated with the mixture of the extracts from the XP cells in two different genetic complementation groups. These results indicate that DNA repair occurred in the cell-free extract similarly to the DNA repair in living cells. We found no effect of p53 protein on a DNA repair mechanism in the cell-free system, although the p53 protein reduced a UV-induced mutation frequency in human cells.Incorporation of [^<32>P] dATP to the unirradiated plasmid pMY189 by DNA replication also occurred when the plasmid was incubated with SV40 T antigen and the extract of normal or XP cells. We found that the extract obtained from S-phase cells has high activity to carry out the DNA replication in the cell-free system.