Induction and processing of promutagenic O4-ethylthymine lesion in specific gene segments of plasmid DNA

High affinity antibodies were used for the quantitative assessment of the miscoding O4-ethylthymine (O4-EtThy) base lesion in nanogram amounts of membrane transblotted restriction fragments of ENU treated DNA. The polyclonal antibody (TB3) specifically recognized attomoles of the alkylation adducts in modified DNA with no cross-reactivity to an excess of unmodified DNA. The sensitivity of the immuno-quantitative method was determined to be in the range of 76 attomoles to 2.43 fmol, corresponding to 0.24 x 10(-7) to 7.9 x 10(-7) adducts per nucleotide in plasmid DNA. Modification levels in ras and tk genes were estimated as 0.025 and 0.014 adducts respectively. Specific antibody binding was proportional to the dose of ENU and size of the DNA fragments. In differentially ethylated ras gene, the amount of O4-EtThy was quantified as 0.026, 0.08 and 0.13 adducts per gene fragment. A DNA concentration dependent antibody binding was observed with large (23.13 and 9.41 kb) and smaller (2.02 kb) fragments of HindIII digested ENU treated phage lambda DNA. To monitor the repair of O4-EtThy lesions in specific segments, damage was assessed in sequences of plasmid DNA established in various Escherichia coli strains. The loss of antibody binding to O4-EtThy adducts in ethylated DNA fragments of 6.4 kb ras gene and 3.6 kb tk gene occurred with an approximate t1/2 of 45 and 35 min, respectively, in the repair proficient wild type E. coli. On the contrary, no repair was seen in the alkyltransferase deficient double mutant ada-ogt- strain. The results specifically demonstrate the sensitivity of the immunological technique and the unique ability of the O4-EtThy specific antibodies to scan this promutagenic base lesion and its repair in very small amounts of selected gene segments in DNA.