DNA Charge Transport Chemistry & Biology

描述（由申请人提供）：本提案旨在表征与DNA介导的电荷传输（CT）相关的化学和生物学。具体而言，DNA CT在氧化应激下DNA如何受损和修复中的作用将被探索。实验提出了在长距离以及DNA介导的信号调节蛋白的DNA氧化损伤检查。我们将研究的距离范围内的氧化损伤是漏斗状的控制序列在线粒体中使用铑光氧化剂产生的损害在遥远的网站。线粒体DNA突变及其与癌症的相关性也将被确定。我们将进一步描绘DNA CT的作用，在DNA损伤检测碱基切除修复（BER）酶，含有4Fe-4S簇。我们将制备一系列BER酶突变体来表征DNA/蛋白质CT的路径。将使用DNA电化学、EPR、瞬态吸收光谱和AFM探测CT。在体外蛋白质氧化鸟嘌呤自由基的DNA将产生的空穴注入拴系金属嵌入剂。E.大肠杆菌将测试两种BER酶MutY和内切酶III（EndoIII）的突变体在检测碱基损伤方面的有效性。将绘制这种生物辅助功能，DNA/蛋白质CT研究，和人类MutY同源物的癌症易感性之间的相关性。DNA介导的转录调控CT也将在soxR中进行检查，soxR是一种来自E. coli中含有Fe-S簇。我们将测试转录的启动从远处通过使用系留的光氧化剂的DNA结合的soxR的远程氧化。除了这些与DNA结合的蛋白质的CT研究，我们将构建DNA组件含有拴系金属嵌入剂，环化的配合物的Ir和Re（CO）二亚胺配合物，表征DNA介导的还原化学与远程DNA介导的氧化。公共卫生相关性：该提案的重点是化学检查当细胞遭受氧化应激时DNA如何受损和修复。受损的DNA，如果不修复，会导致癌变。了解损伤和修复过程对于评估癌症易感性至关重要。

DESCRIPTION (provided by applicant): This proposal aims to characterize the chemistry and biology associated with DNA-mediated charge transport (CT). Specifically, roles for DNA CT in how DNA is damaged and repaired under oxidative stress will be explored. Experiments are proposed to examine oxidative DNA damage at long range as well as DNA-mediated signaling to regulatory proteins. We will examine the distance range over which oxidative damage is funneled to a control sequence in mitochondria using rhodium photooxidants to generate damage at distant sites. Mitochondrial DNA mutations and their correlations with cancers will also be determined. We will further delineate the role of DNA CT in DNA damage detection by base excision repair (BER) enzymes that contain 4Fe-4S clusters. We will prepare a series of BER enzyme mutants to characterize the path for DNA/protein CT. CT will be probed using DNA electrochemistry, EPR, transient absorption spectroscopy and AFM. In vitro protein oxidations by guanine radicals in DNA will be generated by hole injection from tethered metallointercalators. Biological assays of repair in E. coli will test how effectively mutants of two BER enzymes, MutY and Endonuclease III (EndoIII), cooperate in detecting base lesions. Correlations will be drawn between this biological helper function, DNA/protein CT studies, and cancer predispositions in human MutY homologues. DNA-mediated CT for transcriptional regulation will also be examined in soxR, a transcriptional activator from E. coli containing an Fe-S cluster. We will test the initiation of transcription from a distance through long range oxidation of DNA-bound soxR using tethered photooxidants. Paralleling these CT studies with DNA-bound proteins, we will construct DNA assemblies containing tethered metallointercalators, cyclometallated complexes of Ir and Re(CO) diimine complexes, to characterize DNA-mediated reduction chemistry versus long range DNA- mediated oxidations. PUBLIC HEALTH RELEVANCE: This proposal is focused on examining chemically how DNA is damaged and repaired when cells undergo oxidative stress. Damaged DNA, if unrepaired, leads to cancerous transformations. Understanding both the damage and repair process is critical to evaluating cancer predispositions.