One objective of this proposal is to use photochemical methods which we have developed to map carcinogen and mutagen sites on DNA, and to use those data to establish how binding is coupled to DNA structure variation, at one base resolution. We are specifically interested in the microscopic distribution of benzo(a)pyrene dioplexoide (PBDE), nitrosopyrene (NP), ethidium bromide (EB) methylene blue (MB), copper ion (Cu+2), and singlet oxygen (*02) attack sites within gene-sized DNA targets, and within the nucleosome subunits of chromatin. We have focused our attention on two specific DNA regions as models for the coupling between small molecule binding and local variation of DNA secondary structure: the oligo-A segments within kinetoplast DNA, and the 5' end of the human c-myc proto-oncogene. For each, we have shown that DNA structure is best described as a dynamic equilibrium, and propose experiments to test the possibility that carcinogen binding is allosterically coupled to the underlying equilibrium. In a related set of experiments, we have proposed to map the distribution of BPDE and NP binding sites on the supF gene in the mammalian shuttle vector pZ189. It has been determined by others that in a mammalian 293 cell line, BPDE-mediated mutagenesis in supF is targeted to specific sequences. The goal of this subproject is to establish a rigorous relationship between carcinogen binding and site specific mutagenesis in this mammalian assay system. The last major goal of this work is to explore the sequence specific binding of single-strand oligonucleotide to the c-myc gene control region. We have shown that such interactions occur, and result in the formation of a triple strand complex, by a process which may be coupled to the DNA structure equilibrium which we have detected in the myc control region. We propose experiments which will define the structure and specificity of this complex, its coupling to DNA structure change, and its potential as a repressor of myc gene expression.
| Chan, S S; Breslauer, K J; Hogan, M E et al. (1990) Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending. Biochemistry 29:6161-71 |
| Boles, T C; Hogan, M E (1987) DNA structure equilibria in the human c-myc gene. Biochemistry 26:367-76 |
