Diverse Nucleic Acids Chemistry: FundamentalStudies and Applications.

Project Summary Our research group addresses fundamental questions concerning how nucleic acids are damaged and what the consequences of damage are. We also capitalize on the fundamental discoveries made in these investigations to create chemical tools that are useful in studies on cells. To bring these research projects to fruition, we utilize organic chemistry, biochemistry, as well as molecular and cell biology. 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. What is meant by this last statement is that it is well known that many agents that damage DNA are cytotoxic. However, is it DNA damage that is cytotoxic, or is subsequent (bio)chemistry that is responsible cellular effects, including death? Three nucleic acid research topics will be investigated during the requested funding period. Continuing with the theme that DNA damage is not always the end of the story, we will investigate the downstream effects of two forms of DNA damage in bottom-up synthesized nucleosome core particles (NCPs) and in cells. In one research direction we will examine a nonenzymatic covalent modification (NECM) of a histone that we discovered. The NECM is derived from a DNA lesion that was discovered in studies using NCPs and that we recently showed is produced in cells. We will examine the formation of protein cross-links with this modification in NCPs and in cells. The latter will be facilitated by developing molecules that produce the protein modification in cells independent of DNA damage. In another investigation we will study the reactivity of the major deoxyribose oxidation product produced by hydroxyl radical, an important DNA damaging agent produced by g-radiolysis. These studies will also be carried out in NCPs containing the DNA lesion at defined sites and in cells where the lesion is randomly produced. A second research area is inspired by our previous mechanistic studies on DNA damage. Using this knowledge, we plan to synthesize molecules that sensitize cells to g-radiolysis and photodynamic therapy. The molecules are modified nucleotides that will be incorporated into DNA and designed to produce forms of DNA damage that are more deleterious to cells (e.g. DNA-protein cross-links) when exposed to the DNA damaging agents. These experiments will be carried out in NCPs and in cells. Finally, in the spirit of the MIRA program that does not require overlap between projects, we are pursuing a very different research topic, poly(ADP-ribose) (PAR). PARs are transiently formed pyrophosphate nucleic acids of growing interest. The proposed research is motivated by our recent studies involving the design of probes to identify PAR-protein binding interactions. We propose to develop a reliable method for chemically synthesizing PARs of defined length. This chemistry will greatly facilitate studies on PAR structure and molecular recognition carried out by us and others in the scientific community.