SUMOylation and Cell Sensitivity to Top1 Poisons

描述（由申请人提供）：真核DNA拓扑异构酶I（Top1 p）在DNA复制、转录和重组中起重要作用，并通过双链体DNA中单个DNA链的瞬时断裂和重新连接来催化DNA拓扑结构的变化。Top1 p也是喜树碱（CRT）的靶标，其可逆地稳定共价酶-DNA中间体。在S期，复制叉与CRT稳定的复合物的碰撞产生DNA损伤，信号细胞周期停滞和细胞死亡。然而，人们对产生的病变的性质和解决Top1 p诱导的DNA损伤的细胞过程知之甚少。 泛素和泛素样蛋白（如SUMO）的翻译后蛋白质修饰已成为控制细胞周期进程、DNA修复、胞吞作用、核质运输、转录、染色质结构和细胞分裂的关键调节机制。SUMO通过E1、E2（Ubc 9）和在某些情况下E3酶的顺序作用，通过异肽键连接到靶蛋白上。人类Top1 p的SUMO化与核仁定位和CRT敏感性的改变相关。然而，调节Top1 p的SUMO结合的机制和SUMO化对有效修复由靶向Top1 p的化疗药物诱导的DNA损伤所需的细胞途径的功能后果尚未得到解决。 该提案的目标是确定Ubc 9催化的sumoylation在保护细胞免受酿酒酵母中Top1 p毒素的影响中的作用。Top1 p中毒和SUMO结合的高度保守机制，与条件ubc 9 -10突变体的分离相一致，该突变体对Top1 p诱导的损伤具有增强的敏感性，使得这种遗传上易于处理的系统特别适合于SUMO化和Top1 p介导的致死性的研究。还将研究SUMO蛋白酶、Ulp 2 p和Top1 p在维持基因组稳定性方面的遗传相互作用。为了实现这一目标，综合的生化，结构和遗传学分析将评估Ubc 9 p底物特异性的缺陷，揭示这些改变的结构基础，并定义细胞过程，其调节Top1 p毒素抗性的功能受到SUMO结合缺陷的影响。人/酵母Ubc 9 p嵌合体将进一步定义决定底物特异性的残基。进一步的研究将确定是否有类似的机制调节ulp 2Adelta细胞对Top1 p水平的敏感性和基因组稳定性。

DESCRIPTION (provided by applicant): Eukaryotic DNA topoisomerase I (Top1p) plays important roles in DNA replication, transcription and recombination, and catalyzes changes in DNA topology through the transient breakage and rejoining of a single DNA strand in duplex DNA. Top1p is also the target of camptothecin (CRT), which reversibly stabilizes a covalent enzyme-DNA intermediate. During S-phase, replication fork collisions with CRT-stabilized complexes produce DNA lesions that signal cell cycle arrest and cell death. However, little is known about the nature of the lesions produced and the cellular processes that resolve Top1p-induced DNA damage. Post-translational protein modifications by ubiquitin and ubiquitin-like proteins (such as SUMO) have emerged as critical regulatory mechanisms governing cell cycle progression, DNA repair, endocytosis, nucleocytoplasmic trafficking, transcription, chromatin structure and cell division. SUMO is attached to target proteins via an isopeptide linkage by the sequential action of an E1, E2 (Ubc9) and, in some cases, E3 enzymes. Sumoylation of human Top1p has been associated with alterations in nucleolar localization and CRT sensitivity. However, the mechanisms regulating SUMO conjugation of Top1p and the functional consequences of sumoylation on cellular pathways required for the effective repair of DNA lesions induced by chemotherapeutic agents that target Top1p have yet to be addressed. The goal of this proposal is to define the role of Ubc9-catalyzed sumoylation in protecting cells from Top1p poisons in the yeast Saccharomyces cerevisiae. Highly conserved mechanisms of Top1p poisoning and SUMO conjugation, in concert with the isolation of a conditional ubc9-10 mutant with enhanced sensitivity to Top1p-induced damage, makes this genetically tractable system particularly suited to the study of sumoylation and Top1p-mediated lethality. Genetic interactions between the SUMO protease, Ulp2p and Top1p in maintaining genomic stability will also be investigated. To accomplish this, integrated biochemical, structural and genetics analyses will assess the defects in Ubc9p substrate specificity, reveal the structural basis for these alterations and define the cellular processes whose function in modulating resistance to Top1p poisons is affected by defects in SUMO conjugation. Human/yeast Ubc9p chimeras will further define residues that dictate substrate specificity. Additional studies will determine if similar mechanisms regulate ulp2Adelta cell sensitivity to Top1p levels and genomic stability.