SITE-SPECIFIC INDUCTION AND REPAIR OF BENZO[A]PYRENE DIOL EPOXIDE DNA-DAMAGE IN HUMAN H-RAS PROTOONCOGENE AS REVEALED BY RESTRICTION CLEAVAGE INHIBITION

Most genotoxic DNA base modifications localized at key genomic sequenc es constitute the molecular alterations crucial for mutagenesis and tu morigenesis. We have utilized lesion-rendered inhibition of restrictio n endonuclease cleavage for the analysis of site-specific DNA damage i nduced by -anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a] pyrene diol epoxide, anti-BPDE) in human genes. The H-ras protooncogen e and insulin gene sequences were used as targets for modification in vitro and in vivo. Selective induction of individual facultative bands , resulting from covalent modification of the cognate recognition site s, was observed in modified plasmid DNA for a number of restriction nu cleases. The ras gene-specific damage, at the PsrI, BstYI, NotI and Bs tEII recognition sites, was visualized and quantitated in human genomi c DNA adducted by anti-BPDE. Repair of lesions at hexanucleotide seque nces and/or regions surrounding the restriction site, was assessed as a gradual disappearance of facultative bands in DNA from repair-profic ient human fibroblasts exposed to the carcinogen in confluent culture. Efficiency of the PstI site-specific repair was compared at low and h igh levels of initial damage. Higher genotoxic dose caused a decrease in the extent of adduct removal from the bulk DNA, while the specific site of the ras gene was still subject to fast repair. No measurable P srI site-specific repair was detected in the insulin gene. These resul ts show the region-selective induction of bulky anti-BPDE DNA damage i n non-related genomic targets and suggest that repair of these lesions in human cells proceeds with the efficiency tightly controlled at dif ferent levels of initial genotoxic load.