DNA Lesion Bypass in Sulfolobus solfataricus

DNA是一种遗传物质，它赋予每个细胞和有机体独特的特征。DNA的准确复制以及随后从父母传给后代的过程对生命至关重要。一些环境因素，如化学致癌物、紫外线，甚至是自然的细胞过程，都会对我们的DNA造成氧化或物理损害，这可能会导致突变或暂时阻止其复制。事实上，据估计，在每个人类细胞中，每天都有数以万计的DNA受损部位(损伤)。其他生物基因组DNA中的损伤也很普遍。如果不修复，这些损伤可能会抑制DNA的复制，导致细胞死亡或有害的突变。该项目将使用尖端生物物理技术来研究常见的氧化性DNA损伤如何阻止蛋白质准确复制DNA。该项目产生的结果将在分子水平上决定DNA损伤的后果。除了科学上的重要性，该项目还将支持俄亥俄州立大学的本科生和研究生接受科学培训，为他们提供设计和测试科学假说的工具，以及了解科学发现的本质。细胞DNA经常受到内源和外源的损伤，形成无数的DNA损伤部位，从而导致基因组复制过程中细胞DNA复制机制的停滞。DNA复制的一些关键蛋白质是复制型DNA聚合酶和跨损伤合成(TLS)DNA聚合酶，前者合成大部分DNA，后者绕过DNA并将DNA延伸到受损的DNA位点，但往往容易出错。增殖细胞核抗原的过程性增强了复制聚合酶合成长片段DNA的能力，并在DNA损伤部位的聚合酶切换中发挥了关键作用。目前尚不清楚常见的DNA损伤如何影响复制聚合酶和TLS聚合酶在溶液中的构象动力学。此外，还不知道这两种不同类型的聚合酶在病变部位是如何切换的。为了研究第一个机制问题，该项目将利用Förster共振能量转移技术和停流设备--快速混合器--来监测复制聚合酶和TLS聚合酶在与底物结合以及催化过程中的构象动力学。为了阐明聚合酶开关的分子细节，该项目将使用尖端的单分子技术来研究复制聚合酶和TLS聚合酶如何在DNA损伤部位在增殖细胞核抗原的帮助下被开启和关闭。该项目还将为俄亥俄州立大学的本科生和研究生提供在高级酶学领域接受重要科学培训的机会。该项目将为他们提供设计和检验科学假设方面的培训，并为STEM领域的进一步职业发展和教育机会提供支助。

DNA is the genetic material that gives each cell and organism its unique character. The accurate duplication of DNA as well as its subsequent transfer from parent to offspring is critical for life. Some environmental factors such as chemical carcinogens, UV light, or even natural cellular processes can cause oxidative or physical damage to our DNA, which can cause mutations or temporarily block its duplication. In fact, it has been estimated that there are tens of thousands of damaged DNA sites (lesions) in each human cell per day. Lesions in genomic DNA of other organisms are also widespread. If left unrepaired, these lesions can inhibit the duplication of DNA, cause cell death, or detrimental mutations. This project will use cutting-edge biophysical techniques to investigate how a commonly occurring oxidative DNA lesion prevents proteins from accurately duplicating the DNA. The results generated from this project will determine the consequences of DNA damage at the molecular level. In addition to its scientific importance, the project will also support undergraduate and graduate students at The Ohio State University with opportunities to receive scientific training, provide them tools for designing and testing scientific hypotheses, as well as learning about the nature of scientific discovery. Cellular DNA is frequently damaged by both endogenous and exogenous sources to form a myriad of DNA damaged sites, which can stall the cellular DNA replication machinery during genome duplication. Some key proteins for DNA replication are the replicative DNA polymerases, which synthesize the majority of DNA, and translesion synthesis (TLS) DNA polymerases, which bypass and extend DNA across from damaged DNA sites but often in an error-prone way. The processivity factor PCNA enhances the ability of replicative polymerases to synthesize long stretches of DNA and plays a key role in polymerase switching at sites of DNA damage. It is not known how common DNA lesions affect the conformational dynamics of replicative and TLS polymerases in solution. Furthermore, it is not known how these two different types of polymerases are switched at a lesion site. To study the first mechanistic question, the project will utilize Förster resonance energy transfer techniques and a stopped-flow apparatus, a rapid mixer, to monitor the conformational dynamics of a replicative polymerase and a TLS polymerase during binding to substrates, or PCNA, as well as catalysis. To elucidate molecular details of polymerase switching, this project will employ cutting-edge single molecule techniques to investigate how a replicative polymerase and a TLS polymerase are switched on and off at a DNA damaged site with the help of PCNA. The project will also offer undergraduate and graduate students at The Ohio State University opportunities to receive important scientific training in the field of advanced enzymology. The project will provide them training in designing and testing scientific hypotheses as well as support for further career development and educational opportunities in STEM fields.