title: High resolution mapping of UV-induced photoproducts in the Escherichia coli lacI gene. Inefficient repair of the non-transcribed strand correlates with high mutation frequency.
abstract: UV-induced DNA photoproduct formation and repair has been examined at the gene and nucleotide level in Escherichia coli using two newly developed quantitative assays. A multiplex quantitative PCR assay was used to measure photoproduct formation and repair at the gene level in both the constitutive lacI gene and the inducible lacZ gene, simultaneously. Both genes displayed similar photoproduct formation frequencies (0.4 lesions/kb per 100 J/m2). Following a 15 minute recovery period, 36% and 39% of the damage resulting from 100 J/m2 was removed from the lacI and lacZ genes, respectively. Under the growth conditions applied, the lacZ gene was expressed at a very low rate resulting in 0.3% of beta-galactosidase activity as compared to induced cells. A newly developed reiterative primer extension assay has been employed to examine photoproduct formation and repair at the nucleotide level. Analysis of UV-induced DNA photoproducts in the first 184 base-pairs of the lacI gene of genomic E. coli DNA has revealed that photoproducts are induced linearly with dose and the slope is sequence context-dependent. A post-irradiation recovery period revealed differences in the repair efficiency at individual nucleotides. Repair of photoproducts on the transcribed strand was generally twice as efficient as repair of photoproducts on the non-transcribed strand, indicating that strand-specific DNA repair occurs in the constitutively transcribed lacI gene of E. coli. Comparison of the UV-induced DNA photoproduct distribution with an established UV-induced mutation spectrum from wild-type cells revealed that photoproducts form at all mutagenic hotspots. Some sites of low frequency mutations were not observed to be sites of photoproduct formation. However, not all photoproducts appeared to be mutagenic. This was especially true for those on the efficiently repaired transcribed strand. It is hypothesized that the preferential repair of photoproducts on this strand may prevent many of these photoproduct sites from becoming mutagenic hotspots. These data strongly support the hypothesis that mutations arise at inefficiently repaired photoproducts on the nontranscribed strand.
authors.affiliation: Department of Pathology, University of Vermont, Burlington 05405.
authors.forename: D
authors.initials: D
authors.lastname: Chandrasekhar
authors.forename: B
authors.initials: B
authors.lastname: Van Houten
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D007763:Lac Operon; D056968:Lac Repressors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics*; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)