SUMOylation and Cell Sensitivity to Top1 Poisons

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中单链的瞬时断裂和重新连接来催化DNA拓扑的变化。Top1p也是喜树碱(CRT)的靶标，它可逆地稳定一种共价酶-DNA中间体。在S期，复制叉与CRT稳定的复合体发生碰撞，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(如相扑)的翻译后修饰已成为调控细胞周期进程、DNA修复、内吞作用、核质运输、转录、染色质结构和细胞分裂的关键调控机制。SUMO通过一个E1、E2(Ubc9)以及在某些情况下的E3酶的顺序作用，通过异肽键连接到靶蛋白上。人类Top1p的总甲基化与核仁定位和CRT敏感性的改变有关。然而，调控Top1p相扑结合的机制以及针对Top1p的化疗药物诱导的DNA损伤有效修复所需的细胞通路上的相扑甲基化的功能后果尚未得到解决。这项建议的目的是确定Ubc9催化的总甲基化在保护酿酒酵母细胞免受Top1p毒害中的作用。Top1p中毒和相扑接合的高度保守的机制，加上一个对Top1p诱导的损伤具有更高敏感性的条件性ubc9-10突变体的分离，使得这个遗传上易于处理的系统特别适合于SUMOM化和Top1p介导的致死性的研究。相扑蛋白水解酶、Ulp2p和Top1p之间在维持基因组稳定性方面的遗传相互作用也将被研究。为了实现这一点，综合的生化、结构和遗传学分析将评估Ubc9p底物专一性的缺陷，揭示这些变化的结构基础，并确定其调节Top1p毒物抗性的细胞过程受到相扑结合缺陷的影响。人/酵母Ubc9p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类似的机制是否调节ulp2ADelta细胞对Top1p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.