DNA Protein Cross-Links: Metabolic Sources and Repair

PROJECT SUMMARY DNA-protein cross-links (DPCs) form when cellular proteins become irreversibly trapped to DNA as a result of cellular processes such as histone demethylation, lipid peroxidation, and elevation of reactive oxygen species. External and therapeutic agents such as UV light, transition metals, and anti-tumor drugs also induce DPC formation. These bulky lesions interfere with essential DNA-protein interactions necessary for DNA replication, recombination, transcription, and repair. DPC repair appears to be essential for normal cell function, as mice deficient in the DPC protease enzyme, SPRTN, are embryonically lethal. Mutations in SPRTN cause Ruijs-Aalfs syndrome in humans, which is characterized by elevated levels of DPCs, genome instability, accelerated aging, and high risk for cancer. Despite the ubiquitous formation of DPCs and their implication in human disease, the structural identities and repair pathways for this class of adducts are not well-known. DPCs are chemically heterogeneous and the study of such bulky biomolecular conjugates is challenging. There is a critical need to generate new tools and strategies to precisely define how endogenous electrophiles elevated in metabolic diseases contribute to the formation of DPCs and, ultimately, contribute towards initiation and progression of metabolic disorders. The long-term goal of the research in my laboratory is to identify DPCs and repair pathways that may ultimately be used for early detection and risk assessment of metabolic diseases. Over the next five years, this project will test the central hypothesis that the specific molecular signatures of DPCs can be profiled across metabolic states using the multistage fragmentation capability of the mass spectrometer (MS). When combined with specific chemical characterization, this approach will reveal specific DNA-protein interactions relevant to DNA repair. Collectively, these studies will 1) identify metabolites responsible for DPCs during altered cellular metabolism and establish analytical tools to 2) uncover the proteins contributing to DPC formation and 3) identify enzymes participating in DPC repair. This approach is innovative because this study will, for the first time, characterize molecular mechanisms for DPC formation and repair using mass spectrometry-based adductomics technology. This technology is analogous to other -omics methodologies such as metabolomics, proteomics, transcriptomics and genomics which make the study of complex human diseases from a global perspective possible. Expected outcomes: our studies will provide insight into metabolites capable of forming DPCs and provide insight into cellular pathways that contribute to genome instability. An increased understanding of such metabolites lays the foundation for preventative strategies that help limit genomic instability and improve human health.