Spectroscopic and thermodynamic studies of the major DNA adduct formed by the carcinogen 1-nitropyrene

Date of Completion

January 1998

Keywords

Biology, Molecular|Chemistry, Biochemistry|Chemistry, Organic

Degree

Ph.D.

Abstract

A major DNA adduct formed by the carcinogen 1-nitropyrene is N-(deoxyguanosin-8-yl)-1-aminopyrene, dG$\sp{\rm AP}.$ In the research presented here, this adduct was studied by a variety of physical techniques. The monomer, dG$\sp{\rm AP},$ was studied by ultraviolet, fluorescence and circular dichroism spectroscopy. Physical studies were also performed on oligonucleotides containing dG$\sp{\rm AP}.$ Oligonucleotide duplexes were used in optical melting studies as well as spectroscopic studies.^ Fluorescence studies of dG$\sp{\rm AP}$ revealed two distinct emission spectra, one that appeared under basic pH conditions and one present under neutral or acidic conditions. Similar emission spectra were seen for different oligonucleotide duplexes. Duplexes where the pyrene is suspected to be intercalated show exciplex fluorescence and give an emission spectrum similar to that of dG$\sp{\rm AP}$ in base. In these duplexes, there were increased fluorescence yields relative to the single-stranded oligonucleotide. Duplexes with two adducts gave emission spectra similar to that of dG$\sp{\rm AP}$ in acidic or neutral solutions.^ Optical melting studies demonstrated that the adduct has a destabilizing effect on duplex formation. An 11 base oligonucleotide, d(CTCATG$\sp{\rm AP}$ATTCC)-d(GGAATCATGAG) was compared with the parent duplex in melting studies. The adducted duplex gave a slightly lower free energy of helix formation than the parent duplex. In a more extensive study, a 13-mer, d(CCCATCG$\sp{\rm AP}$CTACCC), was annealed to a variety of oligonucleotides which placed different bases opposite to the adduct or lacked the complement to the adduct. These duplexes containing a mismatch or a base deletion serve as models of intermediates in the formation of mutations. Here it was observed that the adduct destabilized the duplex relative to the fully complementary parent duplex, though the modified duplex lacking a base opposite to the adduct was nearly as stable as the fully complementary unmodified duplex. The notable stability of this duplex is consistent with the results of mutagenesis studies. Spectroscopic studies indicated that this sequence likely adopts a conformation in which the pyrene is intercalated and adducted base displaced. The biological implications of such conformations has been discussed. ^

Link to Full Text

Share

COinS