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

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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.