Repair of base alkylation damage in targeted restriction endonuclease sequences of plasmid DNA

Sequence specific ethylation damage and repair of ethyl-adducts in selected restriction endonuclease recognition sites withinp220-ras plasmid DNA was assessed by a modified Southern blotting coupled immunoprobing technique. In situ UV irradiation of DNA in gels clearly ameliorated the immunodetection of minute amounts of facultative fragments generated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate sites. Specific and quantitative localization of induced facultative fragments was achieved in as low as 1 ng of DNA digest corresponding to a peak intensity below 0.1 absorbance unit upon laser scanning. An ENU dose dependent increase in the intensity of representative 7.1 and 7.7 kb facultative fragments was observed as a result of cleavage block atEcoRI (G/ATTC) andBamHI (G/GATCC) restriction endonuclease sites, respectively. To determine the repair in prokaryotic cells, the half-life of repairable alkyl-adducts was assessed in plasmid DNA established in variousEscherichia coli strains as a function of post-treatment incubation time in the recovery medium. The repair is indicated by the gradual disappearance of the 7.1, 7.7, 11.9 and 5.5 kb facultative fragments within the wild-type and mutantE. coli strains. The ethyl-adducts withinEcoRI and BamHI restriction sites were effectively lost from the target DNA in repair-proficientE. coli with an estimated t_1/2 of ∼ 40 min. However, decreased overall rate and at least 2.2-times lesser extent of repair was observed in the repair-deficient (ada⁺ogt^- and (ada^-ogt⁺) cells. No measurable repair was noticed in alkyltransferase defective double mutant (ada^-ogt^-) even after 2 h of post-treatment incubation. The repair of ethyl-adducts atNotI site (GC/GGCCGC) in 5.5 kb facultative fragment occurred at a relatively faster rate (t_1/2 of 27 min) in wild-type bacteria. A 1.5-fold slower repair of ethyl-adducts inBamHI and EcoRI sequences containing G/G and A/G at their cleavage sites was observed compared to C/G inNotI sequence. These results demonstrate the regioselective induction of alkyl-adducts in ethylated DNA and their differential repair inE. coli due to varied efficiency of the repair enzymes for promutagenic DNA base lesions present in different sequence context.