DNA Lesion Bypass in Sulfolobus solfataricus

DNA是赋予每个细胞和有机体独特特征的遗传物质。DNA的精确复制以及随后从父母到后代的转移对生命至关重要。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，都可能对我们的DNA造成氧化或物理损伤，从而导致突变或暂时阻止其复制。事实上，据估计，每天每个人类细胞中都有数万个受损的DNA位点（病变）。其他生物体的基因组DNA损伤也很普遍。如果不进行修复，这些损伤会抑制DNA复制，导致细胞死亡或有害突变。该项目将使用尖端的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。这个项目产生的结果将在分子水平上确定DNA损伤的后果。除了具有重要的科学意义外，该项目还将为俄亥俄州立大学的本科生和研究生提供接受科学培训的机会，为他们提供设计和测试科学假设的工具，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损伤位点，这可以在基因组复制过程中阻止细胞DNA复制机制。DNA复制的一些关键蛋白质是复制性DNA聚合酶，它合成大部分DNA，以及翻译合成（TLS） DNA聚合酶，它绕过并延伸DNA穿过受损的DNA位点，但通常以一种容易出错的方式。增殖因子PCNA增强了复制聚合酶合成长链DNA的能力，并在DNA损伤位点的聚合酶转换中起关键作用。目前尚不清楚常见的DNA损伤如何影响溶液中复制酶和TLS聚合酶的构象动力学。此外，目前尚不清楚这两种不同类型的聚合酶是如何在病变部位切换的。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一个停流装置，一个快速混合器，来监测复制聚合酶和TLS聚合酶在与底物或PCNA结合过程中的构象动力学，以及催化作用。为了阐明聚合酶开关的分子细节，该项目将采用尖端的单分子技术来研究在PCNA的帮助下，复制聚合酶和TLS聚合酶是如何在DNA损伤位点上开启和关闭的。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测试科学假设的培训，并为他们在STEM领域的进一步职业发展和教育机会提供支持。

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.