SITE-SPECIFIC RATES OF EXCISION-REPAIR OF BENZO[A]PYRENE DIOL EPOXIDEADDUCTS IN THE HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE GENE OF HUMAN FIBROBLASTS - CORRELATION WITH MUTATION SPECTRA

When populations of repair-proficient diploid human fibroblasts were t reated with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8 ,9,10-tetrahydrobenzo [a] pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being repli cated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3. However, when the population was treated in early G(1) phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229. No such cell-cycle-dep endent difference in distribution of base substitutions occurred in ex cision-repair-deficient cells. To test whether the increase in the rel ative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of remov al of BPDE adducts from individual sites in exon 3 of the HPRT gene. C ells were treated with 0.5 mu M BPDE in early G(1) phase and harvested immediately or after 10, 20, and 30 hr for repair. The nontranscribed strand of exon 3 was analyzed for the original distribution of adduct s and those remaining after repair, using Escherichia coli UVrABC exci nuclease to excise the adducts and annealing a 5' biotinylated gene-sp ecific primer to the DNA and extending if with Sequenase 2.0 to genera te a blunt end at the site of each cut. A linker was ligated to the bl unt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR and analyzed on a sequencing gel. The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or rema ining at specific sites. The rates of repair at individual sites varie d widely along exon 3 of the HPRT gene and were very slow at nt 212 an d 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots.