title: Repair analysis of promutagenic (+)-anti-BPDE DNA adduct in transcriptionally active sequences of plasmid DNA in Escherichia coli.
abstract: The extent of formation and repair of promutagenic (+)-anti-BPDE-N2-dG in transcriptionally active thymidine kinase (tk) gene insert and vector DNA fragments was assessed in the (+)-anti-BPDE treated plasmid p220-tk within the Escherichia coli hosts of varying repair potential. Polyclonal antibody (BP1), specific for (+)-anti-BPDE DNA adduct, was utilized for quantitative estimation of this bulky lesion in nanograms amounts of membrane transblotted DNA fragments. A carcinogen dose-dependent quantitative antibody binding response, due to selective recognition of the major (+)-anti-BPDE adduct, was seen with various DNA fragments separated by gel electrophoresis. The sensitivity of the immunodetection at 0.2 fmol (+)-anti-BPDE DNA adduct, allowed a linear detection in the range of modification level of 0.64 x 10(-7) to 86 x 10(-7) adducts per nucleotide in plasmid DNA. Based on this sensitivity, detection of 0.07 and 0.46 (+)-anti-BPDE DNA adducts in respective tk and vector DNA fragments was achieved upon immunoanalysis of the in vitro modified DNA. Adduct concentration dependent antibody binding was independent of size of the vector or insert fragments. Antibody binding response, to DNA modified in vivo, was dependent upon the dose of (+/-)-anti-BPDE to plasmid DNA replicating within bacterial hosts. The repair of (+)-anti-BPDE DNA adducts was determined as the loss of antibody binding sites in the specific fragments of plasmid DNA within host E. coli. About 50% of the initial DNA damage was repaired from the individual fragments during 15 min post-incubation in the repair-proficient (wild-type) E. coli cells. Complete adduct removal occurred in approx. 60 min of post-incubation period. A significant (91%) decrease in the survival of mutant (uvrA- recA-) cells was observed at 4 microM (+/-)-anti-BPDE treatment without any reduction in the colony forming units in the wild-type cells. On the contrary, no repair was seen in the excision repair-deficient (uvrA-) E. coli cells. The results indicate (1) the selectivity of the immunological method and the unique ability of the (+)-anti-BPDE specific antibodies to monitor the direct loss of this promutagenic base lesion from the in vivo modified DNA (2) the role of host excision repair pathway in efficient removal of adducts from bacterial genome determines the survival of the bacterial cells and (3) the repair of (+)-anti-BPDE DNA adducts in episomally replicating, transcriptionally active sequences occur at a rapid rate presumably due to the ease of accessibility of repair enzymes to lesions within DNA.
authors.affiliation: Department of Radiobiology, The Ohio State University, Columbus 43210, USA.
authors.forename: J
authors.initials: J
authors.lastname: Musarrat
authors.forename: J
authors.initials: J
authors.lastname: Arezina-Wilson
authors.forename: S
authors.initials: S
authors.lastname: Venkatachalam
authors.forename: A A
authors.initials: AA
authors.lastname: Wani
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism*; D018736:DNA Adducts / Q000276:immunology / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D016296:Mutagenesis; D009153:Mutagens; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D018736:DNA Adducts; D004269:DNA, Bacterial; D009153:Mutagens; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase