DNA Charge Transport in Signaling: In Vitro Studies of the Effect of Signaling Partner Interactions on Enzymatic Activity

点击翻译按钮获取中文摘要

In addition to storing genetic information, the DNA duplex is also capable of acting as a mediator of charge transport (CT) along a DNA strand. DNA CT occurs over long molecular distances, and is highly sensitive to any perturbation in base stacking. Even a single base mismatch is sufficient to inhibit DNA CT. The discovery of the redox state-dependent DNA binding affinity of DNA repair enzymes containing redox-active [4Fe4S] clusters stimulated further research. Fascinatingly, it was then found that DNA CT signaling is critical for the DNA repair activity of these enzymes. Numerous studies support a model in which interprotein signaling via DNA CT is crucial for efficiently coordinating their search for lesions within genomic DNA.Now, the discovery of [4Fe4S] clusters also in replication enzymes and the finding that this cluster can function as redox switch in human DNA primase has raised new questions with broader implications: Can redox-signaling from a distance also influence binding and activity of DNA replication enzymes? Can replication proteins with [4Fe4S] clusters signal one another using DNA CT?To date, little is known about DNA CT-mediated oxidation of replication enzymes. Therefore, in the first part of the proposed research project, a transition metal complex will be tethered to a DNA strand to serve as synthetic signaling partner. Different [4Fe4S] replication enzymes will then be incubated with this modified strand. Upon irradiation, the metal complex serves as photooxidant and can induce oxidation of DNA-bound protein via DNA CT. Using suitable assays, changes in the enzyme’s DNA binding or activity as a result of oxidation can be monitored. These experiments will provide valuable insights into how enzymes are influenced by oxidation of the [4Fe4S] cluster.In the second part, signaling between a protein of interest and a protein partner will be investigated. This protein partner will be oxidized separately and then be added to an activity assay for a protein of interest. The latter is envisioned to be oxidized by the protein partner via DNA CT, resulting in changes in the protein’s affinity to DNA and/or its activity. This will allow simulation of signaling between native protein partners, such as primase and polymerase α, which will contribute to learning how these enzymes coordinate their activities via DNA CT. The investigation of mutations in [4Fe4S] cluster enzymes which render them redox inactive will also be an essential aspect in both project parts. Such mutations have previously been linked to cancer and other diseases, and their precise role is currently unknown.A deeper understanding of how these processes are coordinated is crucial, since the correct replication of genomic DNA is essential for all living organisms. The task of unraveling the role of long-range redox signaling in DNA replication has just begun and has the potential to make major and important contributions to our understanding of cellular function.