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 sequences constitute the molecular alterations crucial or mutagenesis and tumorigenesis. We have utilized lesion-rendered inhibition of restriction endonuclease cleavage for the analysis of site-specific DNA damage induced by (+/-)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, anti-BPDE) in human genes. The H-ras protooncogene 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 sites, was observed in modified plasmid DNA for a number of restriction nucleases. The ras gene-specific damage, at the PstI, BstYI, NotI and BstEII recognition sites, was visualized and quantitated in human genomic DNA adducted by anti-BPDE. Repair of lesions at hexanucleotide sequences and/or regions surrounding the restriction site, was assessed as a gradual disappearance of facultative bands in DNA from repair-proficient human fibroblasts exposed to the carcinogen in confluent culture. Efficiency of the PstI site-specific repair was compared at low and high 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 PstI site-specific repair was detected in the insulin gene. These results show the region-selective induction of bulky anti-BPDE DNA damage in non-related genomic targets and suggest that repair of these lesions in human cells proceeds with the efficiency tightly controlled at different levels of initial genotoxic load.
Denissenko
M F
MF
Department of Radiology, Ohio State University, Columbus 43210, USA.
Venkatachalam
S
S
Ma
Y H
YH
Wani
A A
AA
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D002478:Cells, Cultured; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D018736:DNA Adducts; D004791:Enzyme Inhibitors; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004262:DNA Restriction Enzymes