Date of Award
Doctor of Philosophy
Gerald J. Small
Laser-induced fluorescence line narrowing (FLN) and non-line narrowing (NLN) spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7[beta],8[alpha]-dihydoxy-9[alpha],10[alpha]-epoxy-7,8,9, 10-tetrahydrobenzo (a) pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in the oligonucleotide, 5'-d(CCATCGCTACC) · (GGTAGCGATGG), exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction (~25%) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant [pi]-[pi] stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides d(CTATG1G2G3TATC) were studied as a function of flanking bases. In single stranded form the adduct at G2 or G3 (5'-flanking base guanine) adopts a conformation with strong interaction with the bases. In contrast, the adduct with a 5'-flanking thymine exists in a primarily helix-external conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3'-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N2-dG in DNA isolated from the skin of mice treated topically with benzo[a]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N2-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG. The (+)-cis-adducts were repaired more slowly than most other adducts. Moreover, the (+)-trans-adducts exhibited a broad distribution of base-stacked, partially base-stacked, and helix-external conformations and the adducts with the base-stacked conformation are repaired less readily than the others;Optical and hole burning properties of the DNA binding fluorescent dye TO-PRO-3 were studied using absorption and fluorescence spectroscopy. We found that saturated hole depth of TO-PRO-3 decreased dramatically when bound to DNA. The saturated hole depth and hole growth kinetics of the dye bound to DNA also changed depending on DNA types, sequences and lengths all of which affect the interaction between the dye and DNA. Unlike the saturated hole depth and hole growth kinetics, the hole width of the dye bound to DNA remained practically the same. It is concluded that the dye binds to DNA in various modes, and the binding mode of the dye can be external and partially intercalated. The distribution of the binding modes and the coupling of the dye with its environment are found to be responsible for differences in the observed hole burning properties. The hole growth kinetic property of the homodimeric dye TO-TO-3 also depended on its environment.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Suh, Myungkoo, "Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes " (1995). Retrospective Theses and Dissertations. 11091.