Base-Pairing & Stacking Properties of 8-Hydroxyguanine

Gannett, Peter

Abstract

8-Hydroxydeoxyguanine (8-OHdG) is a DNA base alteration resulting from oxidative damage. This adduct has been shown to result from radiation damage and rom exposure to a variety of chemical carcinogens. Significantly, 8-OHdG preferentially forms in the target organs of certain carcinogens and its incorporation into DNA templates causes mis-reading. These and other data have lead to the proposal that the 8-OHdG, by analogy to other base modifications, are due to changes in both base-pairing and base-stacking properties since conversion of dG to 8-OHdG results in changes in base glycosidic bond angle torsion, 8-OHdG bears a proton on N7, and the imidazole ring's electronic environment is drastically altered. Therefore, the purpose of this proposal is to determine the base-pairing and base-stacking properties of 8-OHdG. The base/sugar geometry and the presence of an N7-proton in 8-OHdG suggest that 8-OHdG may for Hoogsteen base-pairs. To examine this, NMR techniques will be used to study the base-pairing properties of 8-OHdG with deoxycytosine (dC) and mis-matches with deoxyadenonsine (dA) and deoxythymidine (dT). The use of monomers minimizes contributions from base-stacking. In addition, the base-pairing properties of 8- methoxydeoxyguanine (8-OMedG), which has the same base/sugar geometry but no N7-proton, will be examined to assess the importance of the N7-proton of 8-OHdG. Base-stacking interactions will be examined to assess the importance of the N7-proton of 8-OHdG. The usual techniques will be applied and include correlation spectroscopy (COSY), NOE and NOESY 2-D NMR. Alterations in base-stacking are expected due to changes in the electronic environment about the imidazole ring of 8-OHdG. Modification of the base- pairs at adjacent sites and local and/or global geometry may also be observed. These studies will determine the changes in base-pairing and base-stacking of and caused by 8-OHdG relative to dG. Insight into the relationship of these factors and the mutagenic nature of 8-OHdG will be gained. These studies will also provide a foundation for future studies including base- pair """"""""mis-matches"""""""" between 8-OHdG and bases other that dC, the sequence dependence of alterations in base-stacking interactions, base-pairing and base-stacking computation studies. These studies also provide data of general interest regarding the factors which control DNA conformation.