Effect of nuclear environment on the distribution of benzo[a]pyrene diol epoxide-induced adducts in the HPRT gene of human fibroblasts.

(+/-)-7beta,8alpha- Dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]py rene (BPDE) is the principal reactive metabolite of the carcinogenic 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, until recently, investigation of BPDE-induced adduct formation at the nucleotide level in intact mammalian cells has not been feasible. We used ligation-mediated polymerase chain reaction (LMPCR) in conjunction with Escherichia coli UvrABC excinuclease to investigate the distribution of BPDE-induced adducts in the non-transcribed strand of exon 3 of the HPRT gene in normal human fibroblasts at the level of individual nucleotides to single nucleotide resolution using synchronized cell populations. We found that the relative distribution of BPDE adducts in the region of interest was essentially the same in cells treated in early G1 phase, S-phase, late G2/M phase, and in cells blocked at metaphase. Furthermore, for almost all nucleotide positions, the relative distribution of BPDE adducts in the intact cells was very similar to that found when purified DNA was treated with BPDE in vitro. The only exception was that in vivo, adduct formation at a region of six consecutive guanines, i.e. nucleotides 207-212, was strongly enhanced compared with that seen with DNA treated in vitro. No obvious nucleosomal structures or other protein-DNA interaction were detected within the region of interest by in vivo footprinting with micrococcal nuclease and other reagents revealed. In vitro studies mapping BPDE-induced adduct formation using Sequenase and UvrABC excinuclease suggested that this region of six consecutive guanines adopts a special DNA conformation. Therefore, 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 constant throughout the cell cycle.