SUMOylation and Cell Sensitivity to Top1 Poisons

SUMO 化和细胞对 Top1 毒物的敏感性

基本信息

批准号：
7416724
负责人：
MARY-ANN BJORNSTI
金额：
$ 28.09万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
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/7416724
关键词：
Address Affect Binding Biochemical Camptothecin Catalysis Cell Cycle Arrest Cell Cycle Progression Cell Death Cell division Cell physiology Cells Chimera organism Chromatin Structure Class Cleaved cell Codon Nucleotides Complement Complex DNA DNA Damage DNA Repair DNA biosynthesis DNA lesion Defect Drug effect disorder Endocytosis Endopeptidases Enzymes Eukaryotic DNA Topoisomerase I Event Gene Dosage Genetic Genetic Recombination Genetic Transcription Genome Stability Genomics Goals Human In Vitro Lesion Mediating Modification Mutate Mutation Nature Pathway interactions Peptide Hydrolases Pharmaceutical Preparations Phase Play Poison Poisoning Post-Translational Protein Processing Proteins Reaction Reporting Resistance Role Saccharomyces cerevisiae Single-Stranded DNA Site Small Interfering RNA Structure Substrate Specificity Suppressor Mutations System Technology Toxic effect Translating Ubiquitin Ubiquitin Like Proteins Yeasts antitumor agent base chemotherapeutic agent genetic analysis in vivo mutant novel repaired response trafficking ubiquitin-protein ligase

项目摘要

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（由申请人提供）：真核DNA拓扑异构酶I（TOP1P）在DNA复制，转录和重组中起着重要作用，并通过瞬态破裂和重新结合单个DNA链在DUPLEX DNA中的瞬时破裂和重新加入DNA拓扑的变化。 Top1p也是camptothecin（CRT）的靶标，它可逆地稳定共价酶-DNA中间体。在S期间，复制叉与CRT稳定复合物的碰撞会产生信号细胞周期停滞和细胞死亡的DNA病变。但是，对产生的病变的性质以及解决TOP1P诱导的DNA损伤的细胞过程知之甚少。泛素和泛素样蛋白（例如SUMO）的翻译后蛋白质修饰已成为负责细胞周期进展，DNA修复，内吞作用，核细胞胞胞菌素，转录，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）的顺序作用以及在某些情况下是E3酶，通过异肽键连接到靶蛋白。人top1p的sumoylation与核仁定位和CRT敏感性的改变有关。但是，调节TOP1P的相扑结合的机制以及Sumoylation对靶向TOP1P诱导的有效修复DNA病变所需的细胞途径的功能后果，尚未解决。该提案的目的是确定UBC9催化的sumoylation在保护细胞免受酵母菌酿酒酵母中TOP1P毒物免受Top1p毒物免受酿酒酵母的作用。 TOP1P中毒和相扑结合的高度保守的机制与有条件的UBC9-10突变体的隔离，对TOP1P诱导的损伤的敏感性增强，使该遗传障碍系统特别适合于Sumoylation和Top1p介导的top1p介导的研究。在维持基因组稳定性方面，SUMO蛋白酶，ULP2P和TOP1P之间的遗传相互作用也将得到研究。为此，综合的生化，结构和遗传学分析将评估UBC9P底物特异性的缺陷，揭示了这些改变的结构基础，并定义了在调节对TOP1P毒药抗性的功能的细胞过程。人/酵母UBC9P嵌合体将进一步定义决定底物特异性的残基。其他研究将确定相似的机制是否调节ULP2ADELTA细胞对TOP1P水平和基因组稳定性的敏感性。