R15 AREA: Replication in the Presence of Oxidative DNA damage

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DESCRIPTION (provided by applicant): Project Summary Oxidative DNA damage is associated with a range of human disease states involving the progressive loss of developmental and neurological functions, including Cockaynes syndrome, Parkinsons, and Alzhiemers disease. It is by far, the most common form of damage encountered by cells, and it is widely speculated that disease is the result of a gradual accumulation of damage and mutations that eventually compromises cellular function or viability in repair compromised or naturally aged individuals. Yet despite these associations, the cellular mechanism by which oxidative lesions are processed during replication in vivo remains largely uncharacterized. In part, this is because cells devote a suite of enzymes to the repair and tolerance of oxidative damage that appear redundant in biochemical assays and in part because there is a lack of cellular assays that make it challenging to address in mammalian cells The results of this proposal will address these questions directly using the model organism of E.coli, where both replication and oxidative DNA repair are highly conserved. We have established unique cellular assays in E. coli to monitor replication fork processing, and our ability to rapidly purify glycosylases and generate mutants will allow us to directly and definitively determine the how oxidative lesions are processed in vivo. We describe three aims that will be accomplished. 1) Using a unique cellular assay to monitor the repair of lesion in vivo we will identify the biologically relevant oxidative DNA glycosylases that are responsible for the global repair of the genome and subseuqnetly identify the substrate lesions using LC/MS/MS and GC/MS. 2) We will identify the genes and mechanism by which a novel global sensor of oxidative stress transiently shuts downs replication and transcription in response to oxidative stress. 3) We will determine the contribution that repair and translesion synthesis have in processing oxidative lesions encountered during replication and identify the intermediates that arise during the recovery of replication in the presence of these lesions. The results of these studies will allow researchers to determine whether specific oxidative repair deficiencies or impaired processing events lead to human disease states and may suggest novel therapeutic approaches targeting either these replication or repair pathways. PUBLIC HEALTH RELEVANCE: Significance: The results from this project will enhance our understanding of the specific roles that oxidative DNA damage has in causing human disease. Reactive oxygen species are directly or indirectly associated with a range of human hereditary diseases, including Parkinsons, Alzhiemers, amyotrophic lateral sclerosis, Friedreich's ataxia, Fanconi anemia, and Cockayne syndrome. In addition, there is increasing evidence to suggest that reactive oxygen species play a significant role in both spontaneous cancers and the normal aging process. Identifying the cellular role for oxidative repair enzymes and how lesions are processed during replication will allow researchers to examine whether specific repair deficiencies are causative of these human disease states. Furthermore, since oxidative DNA damage generates strong signals for apoptosis, the research may lead to novel modes of chemotherapeutics, involving selective inhibition of repair enzymes identified in this study combined with administration of replicational inhibitors or antioxidants.