In Vivo Regulation and Inhibition of Ribonucleotide Reductase

描述（由申请人提供）：维持足够和平衡的脱氧核糖核苷酸（dNTP）池对于忠实的DNA复制和修复至关重要。失去对dNTP池的正常控制可导致细胞死亡、基因组不稳定和人类癌症易感性。核糖核苷酸还原酶（RNR）的调控主要负责控制细胞dNTP池的相对比例和数量。RNR在dNTP生物合成中的核心作用也使其成为治疗许多恶性肿瘤的成功靶点。RNR酶由两个亚基组成：R1亚基结合四种NDP底物以及控制底物特异性和周转率的变弹性效应物（NTPs和dATP）， R2亚基容纳启动R1核苷酸还原所需的必需酪氨酸自由基。RNR的酶活性可以通过其亚基的变构、转录、蛋白抑制剂关联和亚细胞区隔来调节。出芽酵母已经成为研究RNR活性复杂调控机制的一个典型模型系统。本研究通过结合生物化学、细胞生物学和遗传方法，重点研究控制RNR活性和细胞dNTP池的机制。我们的中心假设是，这些调控机制整合在一起，在不同的生长条件下维持最佳的dNTP池，从而确保高保真的DNA合成和修复。提出了三个具体目标：(1)验证Sml1蛋白通过阻碍R1活性位点的再生来抑制RNR酶的假设，并确定R1-Sml1相互作用和Sml1降解的分子决定因素；(2)研究细胞周期和DNA损伤检查点对R2亚基亚细胞定位的调控；(3)表征新发现的小蛋白Sld1在RNR调控中的作用。Sld1属于一个小蛋白RNR调节因子家族，包括酿酒酵母的Sml1和Sld1， pombe的Spd1，以及其他真菌基因组编码的同源物。重点是获得对这些进化上保守的小尺寸RNR调节蛋白如何控制RNR活性的机制理解。从酵母RNR研究中获得的经验教训将作为理解真核细胞中RNR调控和dNTP池控制的范例，也可能为RNR抑制以及抗肿瘤和抗病毒药物的开发提供新的方法。公共卫生相关性：核糖核苷酸还原酶是一种重要的酶，在所有生物体中提供DNA的构建块。这种酶也是临床治疗人类癌症的靶点。该项目的总体目标是了解这种酶的活性是如何在细胞内被控制的，并找到针对这种酶的药物开发的新策略。

DESCRIPTION (provided by applicant): The maintenance of adequate and balanced deoxyribonucleotide (dNTP) pools is essential for faithful DNA replication and repair. Loss of normal control of the dNTP pools can lead to cell death, genomic instability, and predisposition to cancer in humans. Regulation of ribonucleotide reductase (RNR) is largely responsible for controlling the relative ratios and amounts of the cellular dNTP pools. The central role of RNR in dNTP biosynthesis has also made it a successful target in the treatment of a number of malignancies. The RNR enzyme comprises of two subunits: the R1 subunit binds the four NDP substrates as well as the allosteric effectors (NTPs and dATP) that govern substrate specificity and turnover rate, and the R2 subunit houses the essential tyrosyl radical required to initiate nucleotide reduction in R1. The enzymatic activity of RNR can be modulated by allostery, transcription, protein inhibitor association, and subcellular compartmentation of its subunits. The budding yeast S. cerevisiae has emerged as a prototypical model system with which to investigate the complex mechanisms regulating the RNR activity. This proposal focuses on the mechanisms that control the RNR activity and consequently cellular dNTP pools by using a combination of biochemical, cell biology, and genetic approaches. Our central hypothesis is that these regulatory mechanisms are integrated to maintain optimal dNTP pools under different growth conditions so as to ensure high fidelity DNA synthesis and repair. Three specific aims are proposed: (1) To test the hypothesis that the Sml1 protein inhibits the RNR enzyme by impeding regeneration of the R1 active site and to define molecular determinants of the R1-Sml1 interaction and Sml1 degradation; (2) To examine the regulation of subcellular localization of the R2 subunit by the cell cycle and DNA damage checkpoints; (3) To characterize the role of the newly identified small protein Sld1 in RNR regulation. Sld1 belongs to a family of small protein RNR regulators, including the S. cerevisiae Sml1 and Sld1, the S. pombe Spd1, and their homologs encoded by other fungal genomes. The emphasis is to gain a mechanistic understanding of how the RNR activity is controlled by these evolutionarily conserved small size RNR regulatory proteins. Lessons learned from studies of the yeast RNR will serve as a paradigm for understanding of RNR regulation and dNTP pool control in eukaryotic cells, and may also suggest new approaches for RNR inhibition and for antitumor and antiviral drug development. PUBLIC HEALTH RELEVENCE: Ribonucleotide reductase is an essential enzyme that provides the building blocks for DNA in all organisms. This enzyme is also a proven target of clinical treatment of human cancers. The overall goal of this project is to understand how the activity of this enzyme is controlled inside the cell and finding new strategy for drug development targeting this enzyme.