Nm. King et al., In vitro replication and mutagenesis of a novel reversion vector with selective DNA damage in the supF gene, MUT RES-F M, 476(1-2), 2001, pp. 21-28
Citations number
11
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS
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.