EFFECT OF NUCLEAR ENVIRONMENT ON THE DISTRIBUTION OF BENZO[A]PYRENE DIOL EPOXIDE-INDUCED ADDUCTS IN THE HPRT GENE OF HUMAN FIBROBLASTS

(+/-)-7 beta,8 alpha-Dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahy drobenzo[a]pyrene (BPDE) is the principal reactive metabolite of the c arcinogenic environmental pollutant benzo[a]pyrene, Intensive studies of the distribution of BPDE-induced adduct formation in chromatin DNA compared to that in protein-free DNA have been conducted, However, unt il recently, investigation of BPDE-induced adduct formation at the nuc leotide level in intact mammalian cells has not been feasible, We used ligation-mediated polymerase chain reaction (LMPCR) in conjunction wi th Escherichia coil UvrABC excinuclease to investigate the distributio n of BPDE-induced adducts in the non-transcribed strand of exon 3 of t he HPRT gene in normal human fibroblasts at the level of individual nu cleotides to single nucleotide resolution using synchronized cell popu lations, We found that the relative distribution of BPDE adducts in th e region of interest was essentially the same in cells treated in earl y G1 phase, S-phase, late G2/M phase, and in cells blocked at metaphas e, Furthermore, for almost all nucleotide positions, the relative dist ribution of BPDE adducts in the intact cells was very similar to that found when purified DNA was treated with BPDE in vitro. The only excep tion was that in vivo, adduct formation at a region of six consecutive guanines, i.e. nucleotides 207-212, was strongly enhanced compared wi th that seen with DNA treated in vitro, No obvious nucleosomal structu res or other protein-DNA interaction were detected within the region o f interest by in vivo footprinting with micrococcal nuclease and other reagents revealed, In vitro studies mapping BPDE-induced adduct forma tion using Sequenase and UvrABC excinuclease suggested that this regio n of six consecutive guanines adopts a special DNA conformation, There fore, we conclude that rather than reflecting protein-DNA interaction, the enhanced BPDE-induced adduct formation at nucleotides 207-212 in vivo reflects the impact of the physiological environment in the cell nucleus on the local DNA conformation, and that this effect remains co nstant throughout the cell cycle.