Iron-sulfur clusters have been found in many enzymes essential for DNA replication and repair. Here we propose the characterization of the redox chemistry of these critically important DNA-processing enzymes and how this chemistry may be utilized for long range signaling through DNA charge transport (CT) across the genome. We will use DNA electrochemistry to characterize DNA-bound potentials and DNA CT proficiencies of DNA-processing proteins including DNA polymerase ?, polymerase ? and primase as well as several DNA repair proteins. Experiments will be conducted anaerobically using multiplexed DNA chips. Oxidized vs reduced proteins will be prepared electrochemically and both DNA binding and protein activities will be determined for the proteins with [4Fe4S] clusters in 2+ vs 3+ forms. Mutant proteins that differ with respect to their proficiency in carrying out DNA CT will also be isolated and characterized electrochemically and biochemically, including mutations in human proteins known to promote colon cancer. Once the enzyme signatures for oxidation vs reduction have been established, we will prepare oxidized proteins, either electrochemically or with tethered photooxidants, and use the oxidized protein as a signaling partner for another DNA-bound protein. Assays to determine oxidative signaling will include measurements of replication activity and an AFM assay to measure the localization of CT-proficient [4Fe4S] proteins onto DNA strands with a single base-base mismatch. CT-deficient mutants will be examined in parallel, providing critical controls as well as a means to describe cancerous transformations associated with these mutations. These experiments provide an in vitro model for long-range DNA-mediated signaling within the cell. We will also examine long range-range signaling within Escherichia coli among DNA repair proteins that contain [4Fe4S] clusters. We will utilize several in vivo assays for repair to examine how genetically knocking out one protein affects the activity of another. These genetic experiments should allow the determination of possible networks for DNA signaling among different repair pathways. Complementation with plasmids of both CT-deficient and -proficient mutants will be examined. We will also engineer analogous genomic mutations using CRISPR/Cas9, and CRISPRi will be used to tune protein copy number and explore effects on DNA signaling. We will also examine whether other E. coli proteins involved in DNA- processing contain [4Fe4S] clusters and participate in DNA signaling, including UvrC and topoisomerase I. This work will contribute both to our fundamental understanding of protein/DNA signaling across the genome and the important consideration of DNA CT deficiencies in disease.
This proposal describes fundamental studies to describe how DNA-processing proteins coordinate DNA replication and its repair when the DNA is damaged. Mutations in these processes are associated with cancer and correcting these diseases requires an elucidation of this chemistry.
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