REPAIR ANALYSIS OF PROMUTAGENIC (-ANTI-BPDE DNA ADDUCT IN TRANSCRIPTIONALLY ACTIVE SEQUENCES OF PLASMID DNA IN ESCHERICHIA-COLI())

The extent of formation and repair of promutagenic (+)-anti-BPDE-N-2-d G in transcriptionally active thymidine kinase (tk) gene insert and ve ctor 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, wa s utilized for quantitative estimation of this bulky lesion in nanogra ms amounts of membrane transblotted DNA fragments. A carcinogen dose-d ependent quantitative antibody binding response, due to selective reco gnition of the major(+)-anti-BPDE adduct, was seen with various DNA fr agments separated by gel electrophoresis. The sensitivity of the immun odetection at 0.2 fmol (+)-anti-BPDE DNA adduct, allowed a linear dete ction 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, d etection of 0.07 and 0.46 (+)-anti-BPDE DNA adducts in respective tk a nd vector DNA fragments was achieved upon immunoanalysis of the in vit ro modified DNA. Adduct concentration dependent antibody binding was i ndependent 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 re pair of (+)-anti-BPDE DNA adducts was determined as the loss of antibo dy binding sites in the specific fragments of plasmid DNA within host E, coli. About 50% of the initial DNA damage was repaired from the ind ividual fragments during 15 min post-incubation in the repair-proficie nt (wild-type) E. coli cells. Complete adduct removal occurred in appr ox. 60 min of post-incubation period. A significant (91%) decrease in the survival of mutant (uvrA(-)recA(-)) cells was observed at 4 mu M ( +/-)-anti-BPDE treatment without any reduction in the colony forming u nits in the wild-type cells. On the contrary, no repair was seen in th e excision repair-deficient (uvrA(-)) E. coli cells. The results indic ate(1) the selectivity of the immunological method and the unique abil ity of the (+)-anti-BPDE specific antibodies to monitor the direct los s of this promutagenic base lesion from the in vivo modified DNA (2) t he role of host excision repair pathway in efficient removal of adduct s from bacterial genome determines the survival of the bacterial cells and (3) the repair of(+)-anti-BPDE DNA adducts in episomally replicat ing, transcriptionally active sequences occur at a rapid rate presumab ly due to the ease of accessibility of repair enzymes to lesions withi n DNA.