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

信号传导中的 DNA 电荷传输：信号传导伙伴相互作用对酶活性影响的体外研究

• 批准号: 403201756
• 负责人: Dr. Sandra König
• 金额: --
• 依托单位: Division of Chemistry and Chemical Engineering
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/403201756?language=en
• 关键词: DNA; Charge; Transport; Signaling; Vitro

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.

除了储存遗传信息之外，DNA双链体还能够充当沿DNA链沿着电荷传输（CT）的介体。DNA CT发生在长的分子距离上，并且对碱基堆积中的任何扰动都非常敏感。即使是单个碱基错配也足以抑制DNA CT。含有氧化还原活性[4Fe 4S]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。有趣的是，随后发现DNA CT信号对于这些酶的DNA修复活性至关重要。许多研究支持一种模型，其中通过DNA CT的蛋白质间信号传导对于有效协调基因组DNA内的病变搜索至关重要。现在，在复制酶中发现[4Fe 4S]簇，并且发现该簇可以在人类DNA引发酶中起氧化还原开关的作用，这提出了具有更广泛意义的新问题：来自远处的氧化还原信号也能影响DNA复制酶的结合和活性吗？具有[4Fe 4S]簇的复制蛋白质可以使用DNA CT相互发出信号吗？迄今为止，很少有人知道DNA CT介导的氧化复制酶。因此，在拟议研究项目的第一部分，过渡金属络合物将被拴在DNA链上，作为合成信号伴侣。然后将不同的[4Fe 4S]复制酶与该修饰的链孵育。在照射时，金属络合物充当光氧化剂，并且可以通过DNA CT诱导DNA结合的蛋白质的氧化。使用合适的测定法，可以监测由于氧化导致的酶的DNA结合或活性的变化。这些实验将提供有价值的见解酶是如何影响的[4Fe 4S]簇的氧化。在第二部分中，感兴趣的蛋白质和蛋白质伴侣之间的信号将被调查。该蛋白质伴侣将被单独氧化，然后加入到目的蛋白质的活性测定中。后者被设想为通过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质伴侣之间的信号传导，例如引发酶和聚合酶α，这将有助于了解这些酶如何通过DNA CT协调其活动。在[4Fe 4S]簇酶，使它们的氧化还原活性突变的调查也将是在这两个项目部分的一个重要方面。这些突变以前与癌症和其他疾病有关，它们的确切作用目前尚不清楚。更深入地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。解开长距离氧化还原信号在DNA复制中的作用的任务才刚刚开始，并有可能为我们理解细胞功能做出重大贡献。