D. Chandrasekhar et B. Van Houten, In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in Escherichia coli, MUT RES-F M, 450(1-2), 2000, pp. 19-40
Citations number
49
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS
In vivo formation and repair of the major UV-induced DNA photoproducts, cyc
lobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproduc
ts (6-4 PPs), have been examined at the gene and nucleotide level in Escher
ichia coli. Each type of DNA photoproduct has individually been studied usi
ng photoreactivation and two newly developed assays; the multiplex QPCR ass
ay for damage detection at the gene level and the reiterative primer extens
ion (PE) assay for damage detection at the nucleotide level, In the E. coli
lacI and lacZ genes, CPDs and 6-4 PPs form in a 2:1 ratio, respectively, d
uring UV irradiation. Repair of 6-4 PPs is more efficient than repair of CP
Ds since, on the average, 42% of 6-4 PPs are repaired in both genes in the
first 40 min following 200 J/m(2) UV irradiation, while 1% of CPDs are repa
ired. The location, relative frequency of formation, and efficiency of repa
ir of each type of photoproduct was examined in the first 52 codons of the
E. coli lacI gene at the nucleotide level. Hotspots of formation were found
for each type of lesion. Most photoproducts are at sites where both CPDs a
nd 6-4 PPs are formed. Allowing 40 min of recovery following 200 J/m(2) sho
ws that in vivo repair of 6-4 PPs is about fourfold more efficient than the
repair of CPDs. Comparison of the lesion-specific photoproduct distributio
n of the lad gene with a UV-induced mutation spectrum from wild-type cells
shows that most mutational hotspots are correlated with sites of a majority
of CPD formation. However, 6-4 PPs are also formed at some of these sites
with relatively high frequency. This information, taken together with the o
bservation that 6-4 PPs are repaired faster than CPDs, suggest that the cau
se of mutagenic hotspots in wild-type E. coli is inefficient repair of CPDs
. (C) 2000 Elsevier Science B.V. All rights reserved.