SUMOylation and Cell Sensitivity to Top1 Poisons

SUMO 化和细胞对 Top1 毒物的敏感性

• 批准号: 7087936
• 负责人: MARY-ANN BJORNSTI
• 金额: $ 28.93万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087936
• 关键词: SUMOylation; Cell; Sensitivity; Top1; Poisons

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.

描述（申请人提供）：真核DNA拓扑异构酶I（Top1p）在DNA复制、转录和重组中发挥重要作用，并通过双链DNA中单条DNA链的瞬时断裂和重新连接来催化DNA拓扑结构的变化。 Top1p 也是喜树碱 (CRT) 的靶标，可逆地稳定共价酶-DNA 中间体。在 S 期，复制叉与 CRT 稳定的复合物碰撞会产生 DNA 损伤，发出细胞周期停滞和细胞死亡的信号。然而，人们对所产生的损伤的性质以及解决 Top1p 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）的翻译后蛋白修饰已成为控制细胞周期进程、DNA 修复、内吞作用、核细胞质运输、转录、染色质结构和细胞分裂的关键调节机制。 SUMO 通过 E1、E2 (Ubc9) 和（在某些情况下）E3 酶的连续作用，通过异肽键连接到靶蛋白上。人类 Top1p 的 Sumoylation 与核仁定位和 CRT 敏感性的改变有关。然而，调节 Top1p SUMO 结合的机制以及 SUMO 化对有效修复由针对 Top1p 的化疗药物诱导的 DNA 损伤所需的细胞途径的功能影响尚未得到解决。 该提案的目标是确定 Ubc9 催化的 sumoylation 在保护细胞免受酿酒酵母 Top1p 毒物侵害中的作用。 Top1p 中毒和 SUMO 结合的高度保守机制，与对 Top1p 诱导损伤具有增强敏感性的条件性 ubc9-10 突变体的分离相结合，使得这种遗传上易于处理的系统特别适合研究 sumoylation 和 Top1p 介导的致死率。 SUMO 蛋白酶、Ulp2p 和 Top1p 在维持基因组稳定性方面的遗传相互作用也将得到研究。为了实现这一目标，综合生化、结构和遗传学分析将评估 Ubc9p 底物特异性的缺陷，揭示这些改变的结构基础，并定义其调节 Top1p 毒物抗性的功能受到 SUMO 结合缺陷影响的细胞过程。人/酵母 Ubc9p 嵌合体将进一步定义决定底物特异性的残基。其他研究将确定类似的机制是否调节 ulp2Adelta 细胞对 Top1p 水平和基因组稳定性的敏感性。