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）：真核生物DNA拓扑异构酶I （Top1p）在DNA复制、转录和重组中发挥重要作用，通过双链DNA单链的瞬间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，喜树碱可以可逆地稳定共价酶- dna中间体。在s期，复制叉与crt稳定的复合物碰撞产生DNA损伤，这是细胞周期停滞和细胞死亡的信号。然而，对于所产生的病变的性质和解决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E3酶的顺序作用，通过异肽链连接到靶蛋白上。人类Top1p的sumo化与核仁定位和CRT敏感性的改变有关。然而，调节Top1p的SUMO偶联的机制以及SUMO化对有效修复由靶向Top1p的化疗药物诱导的DNA损伤所需的细胞通路的功能后果尚未得到解决。本提案的目的是确定ubc9催化的summoylation在酵母中保护细胞免受Top1p毒害的作用。Top1p中毒和SUMO偶联的高度保守机制，以及对Top1p诱导损伤敏感性增强的条件ubc9-10突变体的分离，使这个遗传上易于处理的系统特别适合于研究SUMO化和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被研究。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SUMO偶联缺陷影响调节对Top1p毒物抗性的细胞过程。人/酵母Ubc9p嵌合体将进一步确定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ulp2Adelta细胞对Top1p水平的敏感性和基因组稳定性。