This Program Project interactively uses organic synthesis, structural biology, and molecular biology to elucidate processes by which bifunctional alkylating adducts degrade DNA replication and repair at a molecular level. Effort will focus on agents in which the two sites of possible nucleophilic attack by the DNA are separated by either two or three carbon atoms. Examples of the former are vinyl chloride metabolites chlorooxirane and chloroacetaldehyde. Examples of the latter are the alpha, beta-unsaturated aldehydes such as acrolein, crotonaldehyde, and hydroxynonenal. The overall goal is to understand how site-specific adducts of these agents perturb DNA structure, and how such structural modifications modulate the processing of genetic information. These agents react with endocyclic and exocyclic nitrogen atoms in DNA bases to form monodentate adducts, cyclic adducts, DNA crosslinks, and perhaps DNA-protein crosslinks. Adducts involving N1 and N2 of deoxyguanosine, N2 and N6 of deoxyadenosine , and N3 and N4 of deoxycytosine will be examined. Cyclic adducts can be formed in two basic configurations due to the fact that the first nucleophilic attack can potentially occur at either reactive site in the bis-electrophile. The resulting """"""""distal"""""""" and """"""""proximal"""""""" adducts have different chemistry and the investigators propose that these chemical differences will be reflected in their three-dimensional structures in duplex DNA, and their biological processing. In this Project, Dr. Harris will develop synthetic methodology for preparation of cyclic adducts and crosslinked species and will explore the chemistry of these adducts so that limitations of time, temperature, pH, etc. can be established. A DNA Synthesis Core Facility directed by Dr. Rizzo interacts closely with the Project. The core facility will establish protocols for the large-scale preparation and purification of adducted oligonucleotides, and provide site-specific adducts for structural and biological studies. In the second Project, Dr. Lloyd will examine how specific adducts affect the processing of genetic information and relate this to chemical and structural differences in the adducts. In the third Project, Dr. Stone will focus on the three-dimensional structures of site-specifically modified oligodeoxynucleotides, using high field NMR spectroscopy, and relating this information to the chemical and biological properties of the adducts. By identifying how adduct chemistry is related to structure and biology in DNA, the investigators' studies are designed to unravel a complex spectrum of genotoxic responses to these bifunctional alkylating agents.
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