Analysis of site-specific binding of (+/-)-anti-benzo[a]pyrene diol epoxide to restriction fragments of pBR322 DNA via photochemical mapping

The binding sites and relative reactivity of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] covalently bound to restriction fragments of pBR322 DNA are determined. (+/-)-anti-BPDE-modified DNA undergoes a photodissociation at the site of these adducts when irradiated with 355-nm laser light, resulting in a scission of the DNA sugar-phosphate backbone producing DNA fragments similar to those of Maxam-Gilbert sequencing reactions. The binding sites of (+/-)-anti-BPDE with each DNA base are determined by sequencing gel analysis of the BPDE-mediated photolysis and laser densitometry of the resulting banding patterns. This technique was used to analyze the binding of (+/-)-anti-BPDE to the 5' and 3' strands of the EcoRI/EcoRV and BamHI/SalI restriction fragments of pBR322 DNA. The reactivity of (+/-)-anti-BPDE to guanine bases within guanine-rich regions of DNA is enhanced by as much as a factor of 17 relative to the least reactive guanines which are flanked by non-guanine bases. The results also show enhanced photocleavage of the backbone corresponding to non-guanine bases in guanine-rich regions. These results suggest either that non-guanine basis in guanine-rich regions are more reactive than identical bases in other regions of the restriction fragment or that photocleavage of the backbone occurs adjacent to a BPDE-modified guanine. The binding profiles of (+/-)-anti-BPDE to pBR322 DNA at a binding density of 0.52 and 0.93 BPDE adduct per strand gave essentially identical binding patterns.(ABSTRACT TRUNCATED AT 250 WORDS)