Biological effects of bulky N7- and C8-deoxyguanosine adducts by a site-specific approach

Compounds that have the ability to alkylate DNA, resulting in the covalent modification of DNA, may induce genetic mutations. Prior studies on the C8-deoxyguanosine (C8-dG) adducts of N-acetyl-2-aminofluorene (AAF), 2-aminofluorene (AF), and 1-aminopyrene (AP), indicate notable similarities in structures, in spite of their different biological effects. However, the biological effects of these adducts have not been compared within the same DNA sequence in cell culture. Also, the major metabolite of the antitumor antibiotic mitomycin C (MC), 2,7-diaminomitosene (2,7-DAM), has the ability to form monoadducts with DNA. In the work described here, comparative mutagenicity of the C8-dG adducts, and the possible mutagenic and cytotoxic effects of the 2,7-DAM-N7-Gua adduct was studied in both bacterial and mammalian cells.

Effects of DNA Adduct Structure and Sequence Context on Strand Opening of Repair Intermediates and Incision by UvrABC Nuclease

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N 2-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed

Interactions of human replication protein A with single-stranded DNA adducts

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N -acetyl-2-aminofluorene-, 1-aminopyrene-, BPDE (benzo[ a ]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)- cis -anti-BPDE-dG, we demonstrated that, on binding of RPA, the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8–9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases