Investigator: Deyu Li, University of Rhode Island

Mentor: Bingfang Yan, University of Rhode Island

Scientific Theme: Molecular Toxicology and Cancer

Abstract: Nuclear and mitochondrial DNA is damaged by endogenous and exogenous insults. DNA adducts arising from nucleic acid damage may cause the development and acceleration of cancer. The AlkB proteins, a group of Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases, have been established to repair DNA alkyl lesions. Nine AlkB homologs exist in mammalian cells (ABH1-8 and FTO). In humans, ABH2 and ABH3 have been identified as DNA repair enzyme. Recently, studies demonstrated that Fe(II)/αKG-dependent enzymes can be inhibited by 2-hydroxyglutarate (2HG) via competitive binding to the Fe(II) center and replacing αKG. 2HG, an oncometabolite, is generated by mutated isocitrate dehydrogenase 1 (IDH1) and IDH2. Cancer-associated IDH mutations alter these enzymes such that the natural αKG is diminished, while the structurally similar 2HG accumulates to high levels within cells. Similar to IDH mutations, fumarate hydratase and succinate dehydrogenase are mutated in human cancers, leading to accumulation of their substrates, fumarate and succinate, respectively. Fumarate and succinate are also competitive inhibitors of αKG-dependent dioxygenases. The central hypothesis of this project is that those oncometabolites inhibit AlkB/ABH DNA repair capacity by replacing the α-ketoglutarate in the catalytic center. The first goal of this project is to characterize the kinetic parameters of oncometabolites on the inhibition of repairing alkyl DNA adducts. We will first synthesize oligonucleotides that contain the proposed DNA adducts, then purify the three AlkB family enzymes, and measure the kcat and Km parameters of αKG and Ki of the oncometabolites in the repair reaction. The second goal is to characterize the cellular inhibitory property of toxic metabolites on the repair enzymes. We will test metabolites’ intervention of DNA repair in both bacteria and mammalian cells through replication efficiency and mutagenicity assays. Overall, these studies will characterize novel cellular targets of oncometabolites and provide a molecular rationale for the inhibition of DNA repair. Once again, DNA damage is a primary initiator of different tumors and completion of the proposed studies will have direct relevance to human health.

Human Health Relevance: DNA damage is the primary cause for many types of cancer. DNA repair enzymes provide the protection against DNA damage. This application will study how the oncometabolites in cellular energy metabolism inhibit repair enzymes and contribute to the process leading to cancer.