REPAIR OF BASE ALKYLATION DAMAGE IN TARGETED RESTRICTION-ENDONUCLEASESEQUENCES OF PLASMID DNA

Sequence specific ethylation damage and repair of ethyl-adducts in sel ected restriction endonuclease recognition sites within p220-ras plasm id DNA was assessed by a modified Southern blotting coupled immunoprob ing technique. In situ UV irradiation of DNA in gels clearly ameliorat ed the immunodetection of minute amounts of facultative fragments gene rated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate sites. Specific and quantitative localiza tion of induced facultative fragments was achieved in as low as 1 ng o f DNA digest corresponding to a peak intensity below 0.1 absorbance un it upon laser scanning. An ENU dose dependent increase in the intensit y of representative 7.1 and 7.7 kb facultative fragments was observed as a result of cleavage block at EcoRI (G/ATTC) and BamHI (G/GATCC) re striction endonuclease sites, respectively. To determine the repair in prokaryotic cells, the half-life of repairable alkyl-adducts was asse ssed in plasmid DNA established in various Escherichia 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 mutant E. coli strains. The ethyl-adducts within EcoRI and BamHI restriction sites were effectively lost from the target DNA in repair-proficient E . coli with an estimated t(1/2) of similar to 40 min. However, decreas ed overall rate and at least 2.2-times lesser extent of repair was obs erved in the repair-deficient (ada(+) ogt(-)) and (ada(-) ogt(+)) Cell s. No measurable repair was noticed in alkyltransferase defective doub le mutant (ada(-)ogf(-)) even after 2 h of post-treatment incubation. The repair of ethyl-adducts at NotI site (GC/GGCCGC) in 5.5 kb faculta tive 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 in Ba mHI and EcoRI sequences containing G/G and A/G at their cleavage sites was observed compared to C/G in NotI sequence. These results demonstr ate the regioselective induction of alkyl-adducts in ethylated DNA and their differential repair in E. coli due to varied efficiency of the repair enzymes for promutagenic DNA base lesions present in different sequence context.