In vitro replication and mutagenesis of a novel reversion vector with selective DNA damage in the supF gene

The ability to detect the most common type of UV-induced mutation, the C to T transition, at the previously characterized hotspot at position 99 of th e supF gene has been demonstrated in a selectively irradiated reversion vec tor, pLS189(Rev). The supF region was amplified, irradiated with 500 J/m(2) UVC or unirradiated, and ligated into the pLS 189(Rev) plasmid. A portion of ligated product plasmid containing the irradiated fragment was sensitive to nicking by T4 endonuclease V, indicating the presence of the most commo n type of UV-induced damage, the pyrimidine dimer. Plasmid containing the i rradiated or unirradiated supF gene was replicated completely in cellular e xtracts from either HeLa or XP-A cells in vitro. Plasmid containing the irr adiated supF gene showed an inhibition of total replication to a level simi lar to those of previous studies with plasmid molecules exposed in their en tirety to 40 J/m(2). Replication of selectively irradiated plasmid resulted in an average reversion frequency of 0.071% in the two extracts; a 42-fold increase over the average spontaneous reversion frequency of unirradiated plasmid. The reversion frequencies were not significantly different between extracts prepared from HeLa and XP-A cells, indicating that neither the re pair status of the cell lines nor the XPA protein itself affect the frequen cy of C to T transitions at position 99 of the supF gene in plasmid replica ted in vitro. These data indicate that the plasmid pSL189(Rev), containing the selectively UV-irradiated SupF gene, is a useful and sensitive tool to study mutagenesis at a specific site. This approach may be applicable to th e investigation of other environmental DNA-damaging agents, by allowing the target gene to be selectively damaged while maintaining the ability of the plasmid to replicate completely. Such a system, amenable to biochemical ma nipulation, may be very valuable in elucidating the function of novel prote ins in the process of mutagenesis. (C) 2001 Elsevier Science B.V. All right s reserved.