A novel approach for analyzing the structure of DNA modified by benzo[alpha]pyrene diol epoxide at single-molecule resolution

Benzo[a]pyrene diol epoxide (BPDE) has been shown to bind specifically to t he exocyclic amino group of deoxyguanosine in duplex DNA. Interestingly, th is metabolite exhibits stereoselectivity in its tumorigenic and mutagenic e ffects. It is thought that local DNA conformation is altered at the site of the adduct, resulting in aberrant biological processes, and that in certai n sequence contexts BPDE-DNA adducts induce bends in the DNA. In the work p resented here, we compared DNA structural alterations of BPDE-modified DNA and unmodified DNA via tapping mode atomic force microscopy (AFM). DNA frag ments 366 base pairs (bp) in length were generated by PCR from the duplicat ed multiple-cloning site of pBEND2 inserted into pGEM-3Zf(-), and either mo ck-modified or treated with BPDE to give modification levels between 1 and 5% of the nucleotides. Control or BPDE-modified DNA was adsorbed to mica an d visualized in air by AFM. The contour lengths and end-to-end lengths of i ndividual molecules were measured. The ratio of end-to-end distance to cont our length was significantly smaller for modified DNA molecules than for th e unmodified DNA preparation, although the frequency distributions of the c ontour lengths were similar for the two preparations. This suggests BPDE-DN A adducts cause significant bending of DNA molecules, confirming previous c onclusions based on more indirect measurements. The average induced bend an gle for BPDE-DNA adducts is estimated to be at least 30 degrees.