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

除了存储遗传信息外，DNA双链还能够作为沿DNA链的电荷传输（CT）的介质。DNA CT发生在很长的分子距离上，并且对碱基堆叠中的任何扰动都高度敏感。即使单个碱基不匹配也足以抑制DNA CT。含有氧化还原活性[4Fe4S]簇的DNA修复酶的氧化还原状态依赖性DNA结合亲和力的发现刺激了进一步的研究。令人着迷的是，随后发现DNA CT信号对于这些酶的DNA修复活性至关重要。许多研究支持这样一种模型，即通过DNA CT的蛋白间信号传导对于有效协调对基因组DNA内病变的搜索至关重要。现在，在复制酶中也发现了[4Fe4S]簇，并且发现该簇可以作为人类DNA引物酶的氧化还原开关，这提出了具有更广泛意义的新问题：远程氧化还原信号是否也会影响DNA复制酶的结合和活性？带有[4Fe4S]簇的复制蛋白能否通过DNA CT相互传递信号？迄今为止，对DNA ct介导的复制酶氧化知之甚少。因此，在提议的研究项目的第一部分，过渡金属配合物将被拴在DNA链上，作为合成信号伙伴。然后将不同的[4Fe4S]复制酶与该修饰链一起孵育。辐照后，金属配合物作为光氧化剂，可通过DNA CT诱导DNA结合蛋白氧化。使用合适的测定方法，可以监测氧化引起的酶的DNA结合或活性的变化。这些实验将为[4Fe4S]簇氧化如何影响酶提供有价值的见解。在第二部分，感兴趣的蛋白质和蛋白质伴侣之间的信号将被研究。该蛋白质伴侣将被单独氧化，然后添加到感兴趣的蛋白质的活性测定中。后者被设想通过DNA CT被蛋白质伴侣氧化，导致蛋白质对DNA的亲和力和/或其活性的变化。这将允许模拟天然蛋白伙伴之间的信号传导，如引物酶和聚合酶α，这将有助于通过DNA CT了解这些酶如何协调它们的活动。研究使[4Fe4S]簇酶氧化还原失活性的突变也将是这两个项目的重要方面。这种突变以前被认为与癌症和其他疾病有关，但它们的确切作用目前尚不清楚。深入了解这些过程是如何协调的是至关重要的，因为基因组DNA的正确复制对所有生物体都是必不可少的。揭示远程氧化还原信号在DNA复制中的作用的任务才刚刚开始，并且有可能为我们对细胞功能的理解做出重大贡献。

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.