Our research group addresses fundamental questions concerning how nucleic acids are damaged and what the biochemical consequences of damage are. We also capitalize on the fundamental discoveries made in these investigations to create enzyme inhibitors, radiosensitizing agents, and tools that are useful in biotechnology. To bring these research projects to fruition, we utilize organic chemistry, biochemistry, as well as molecular and cell biology. Over more than two decades, this research approach has enabled us to uncover novel pathways of DNA damage, adjudicate mechanistic controversies, and reveal biochemical effects of damaged DNA that illustrate that nucleic acid damage itself is not always the end of the story. We request support to continue all 3 aspects of this research program. We will utilize our ability to independently generate reactive intermediates to elucidate questions concerning oxidative damage in free and nucleosomal DNA. For instance, we will examine the reactivity of nitrogen radicals, which we demonstrated are capable of initiating tandem lesion formation via hydrogen atom abstraction, unlike most carbon radicals. Tandem lesions are a deleterious form of DNA damage that are a hallmark of g-radiolysis. Some of the nitrogen radicals are also chameleon-like in that their pKa's are sufficiently high that reasonable quantities of the respective radical cations are present at neutral pH. Radical cations are important species produced from the direct effect of ionizing radiation and initiate hole transfer in DNA. We will study hole transfer in nucleosomal DNA by independently generating radical cations in nucleosome core particles (NCPs) at defined sites. This will enable us to determine the effects of NCP structure on hole migration, a topic that is of increasing interest due to the realization that hole transfer is important in signaling between proteins and DNA. Efforts on understanding the effects of DNA damage will focus on chemistry in NCPs and the consequences of DNA damage-induced histone modification. We will build upon our discoveries that alkylated DNA forms DNA-protein cross-links (DPCs) with histones and that histone catalyzed chemistry of oxidized abasic sites results in modification of lysine residues. These studies will range from experiments in test tubes to cells to determine the prevalence of histone modifications formed in cells and to identify their biochemical (downstream) effects. We will also determine whether DPC formation occurs in NCPs when DNA is alkylated in the minor groove. Finally, we will utilize halogenated purines to potentiate the effects of DNA alkylation by stabilizing the DPCs formed. This research will contribute to our fundamental understanding of DNA damage and its connection to the etiology and treatment of disease.
This proposal describes the elucidation of how DNA damage is formed, what the consequences of damage are, and provide the inspiration for the design of agents that are useful tools. DNA damage is associated with the etiology and treatment of disease, such as cancer. Revealing the molecular basis for such diseases and their treatments is facilitated by understanding the underlying chemistry, and impacts human health.
