Crosslinking Ribonucleotide Reductase Subunits to Study PCET Within

描述（由申请人提供）：核糖核苷酸还原酶（RNRs）负责将核糖核苷酸（ndp）还原为脱氧核糖核苷酸（dndp），并且在所有生物体中都是必需的，以便制造用于DNA复制和修复的单体前体。本研究计划概述了一类RNR结构的发展，其中两个亚基或一个亚基模拟物是共价连接的。在R1和R2亚基中分别加入叠氮基和炔基功能，允许它们通过[3+2]环加成进行位点特异性附着。亚基的共价附着会降低被认为参与PCET途径的氨基酸残基的构象灵活性。沿着提议的PCET途径的氨基酸的这种构象限制和系统突变将促进自由基物种在构建中的建立，从而允许提议的自由基中间体被光谱观察到。观察通路中每个残基的自由基种类将证明它们各自参与脱氧核糖核苷酸还原机制，并允许确定这些PCET事件的速率。对这些速率的温度和同位素依赖性的研究将确定这些过程是否受到蛋白质构象变化或PCET过程本身的限制。本研究的成功完成将对核糖核苷酸还原酶中的自由基转运机制提供详细的机制理解。将哺乳动物细胞中的核糖核苷酸减少机制与细菌和病毒细胞中的核糖核苷酸减少机制进行比较，将为开发能够特异性靶向细菌和病毒RNR中基于自由基的中间体的药物提供见解，从而在不破坏类似哺乳动物过程的情况下抑制其复制和修复。

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) are responsible for reducing ribonucleotides (NDPs) to deoxyribonucleotides (dNDPs) and are required in all organisms in order to make the monomeric precursors used in DNA replication and repair. This research proposal outlines the development of class I RNR constructs in which the two subunits or a subunit mimic are covalently attached. Incorporation of azido and alkynyl functionality into to R1 and R2 subunits, respectively, allows for their site-specific attachment via a [3+2] cycloaddition. The covalent attachment of the subunits will reduce the conformational flexibility of the amino acid residues believed to be involved in the PCET pathway. This conformation restriction and systematic mutation of the amino acids along the proposed PCET pathway will facilitate the build up of radical species in the construct thereby allowing the proposed radical intermediates to be observed spectroscopically. Observation of the radical species of each of the residues in the pathway will prove their respective involvement in the mechanism of deoxyribonucleotide reduction and allow for the rates of these PCET events to be determined. Studies of the temperature and isotopic dependence of these rates will determine if these processes are limited by protein conformational changes or the PCET process itself. The successful completion of the research outlined in this proposal will provide a detailed mechanistic understanding of the radical transport mechanism in ribonucleotide reductase. PUBLIC HEALTH RELEVANCE Comparing the mechanism of ribonucleotide reduction in mammalian cells to that in bacterial and viral cells will provide insight into the development of drugs that can specifically target the radical-based intermediates in the bacterial and viral RNR, thereby inhibiting their replication and repair without disrupting the analogous mammalian processes.