OXIDATIVE DNA DAMAGE AND EXOCYCLIC DNA ADDUCTS

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This application focuses on structural alignments at the DNA duplex level associated with abasic sites, exocyclic adducts, 8-oxo-purine lesions and aromatic amine adducts which play a critical role in chemical and radiation induced carcinogenesis. The solution structure of these lesions will be defined by a combination of NMR and molecular dynamics refinements in defined sequence contexts. New directions in our abasic site research will focus on abasic sites containing deletions opposite the lesion and bistrand abasic site lesions which are refractory to repair. These structural studies will be extended to ribonolactone abasic sites both as isolated lesions and as bistrand lesions relevant to neocarcinostatin action. The exocyclic adduct research will be extended to etheno exocyclic adducts of the potent carcinogen vinyl chloride with deoxyguanine, deoxyadenine and deoxycytidine. We shall probe the generality of syn alignments at the exocyclic adduct and the extent of structural perturbations necessary to accommodate the exocyclic ring within the helix. The structural research will be correlated with research on DNA glycosylases which selectively recognize these exocyclic adducts depending on the base opposite the lesion site. We plan a comparative structural investigation of oxidative damage at deoxyguanine and deoxyadenine in the same sequence context in an attempt to understand the origin of the enhanced mutagenicity of 8-oxo-Dg in contrast to 8-oxo- Da which is non-mutagenic. An attempt will be also made to characterize imidazole ring-opened FAPY adducts of deoxypurines at the DNA oligomer level. Our previous structural research on base substitution alignments at C8-deoxyguanine aromatic amine adduct sites are being extended to single and double deletion frame-shifts in specific sequence contexts established from in vitro replication studies. Our efforts will attempt to define the conformation of (AAF)Dg and (AF) Dg at the frame-shift site and identify the structural differences associated with a single and double deletions. Such a comparative analysis will also be extended to the anti-oxidant carcinogen (ABP)Dg and the food toxin (PhIP)Dg adducts to evaluate contributions from the aromatic amine ring system to structural alignments at the lesion site. The overall goal is to couple our NMR structural investigations with related calorimetric measurements in Prof. Ken Breslauer's laboratory on these four families of DNA lesions prepared in Prof. Francis Johnson's laboratory. These studies will provide the structural-thermodynamics framework necessary for interpretation of mutagenesis experiments in the laboratories of Profs. Arthur Grollman and John Essigmann.