PROTEOMICS OF TOPOISOMERASE-DNA CLEAVAGE COMPLEXES

拓扑异构酶-DNA 切割复合物的蛋白质组学

• 批准号: 6775471
• 负责人: ROBERT M SNAPKA
• 金额: $ 23.55万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6775471
• 关键词: DNA damage; DNA repair; DNA replication origin; DNA topoisomerases; biotechnology; camptothecin; chemical cleavage; crosslink; enzyme structure; isozymes; pharmacokinetics; phosphoester ligase; phosphorylation; posttranslational modifications; protein purification; proteomics; ubiquitin

DESCRIPTION (provided by applicant): We have developed chromosomal cross-link analysis (CXLA) technology for the rapid purification of proteins covalently attached to DNA. CXLA produces proteins in amounts and purity sufficient for unambiguous identification of the proteins and their post-translational modifications by proteomic methods. We will use CXLA together with biochemical and molecular genetic methods to test several hypotheses and models concerning the structure and functions of post-translational modifications of topoisomerases that result from their targeting by anticancer drugs and treatments. Specific Aim I will test the hypothesis that sumoylation of topoisomerase I is involved in detection, signaling and/or processing of topoisomerase I mediated DNA damage by camptothecins. Models for the specific function of sumoylation will be tested and alternate hypotheses will be tested if necessary. Specific Aim II will test the hypothesis that ubiquitin family modifications of topoisomerase II and their function vary depending on the isozyme, its function and the anticancer drugs stabilizing topo II-DNA cleavage complexes. Specific Aim III will test several hypotheses for functions of topoisomerase phosphorylation: (1), That phosphorylation at specific sites is required for DNA cleavage (2), that ubiquitination and sumoylation are driven by phosphorylation at specific sites (3), that prolyl isomerization is required for sumoylation of topoisomerases in drug-stabilized DNA cleavage complexes. Specific Aim IV will test the hypotheses that (1) repair enzymes known from lower phyla, but not yet discovered in humans, are present at sites of topoisomerase mediated DNA damage (2) that different sets of repair enzymes are recruited to replication forks damaged by topo I poisons, topo II poisons and hydroxyurea as a result of differences in fork damage caused by these agents. Studies under Aim IV are also expected to yield new information on the sequence and kinetics of recruitment of repair enzymes to damaged forks, and information on direct interactions of repair proteins and steps in fork repair.

描述（由申请人提供）：我们开发了染色体交联分析（CXLA）技术，用于快速纯化与DNA的共同附着的蛋白质。 CXLA产生的蛋白质和纯度足以通过蛋白质组学方法明确鉴定蛋白质及其翻译后修饰。我们将使用CXLA以及生化和分子遗传学方法来测试有关拓扑异构酶翻译后修饰的结构和功能的几种假设和模型，这些假设和功能是由抗癌药物和治疗靶向的。具体目的我将检验以下假设：拓扑异构酶I的sumoylation参与检测，信号传导和/或加工拓扑素介导的DNA损伤。将测试用于Sumoylation的特定功能的模型，并在必要时测试其他假设。特定的目标II将检验以下假设：拓扑异构酶II及其功能的泛素家族修饰取决于同工酶，其功能和抗癌药物稳定topo II-DNA裂解络合物的抗癌药物。 Specific Aim III will test several hypotheses for functions of topoisomerase phosphorylation: (1), That phosphorylation at specific sites is required for DNA cleavage (2), that ubiquitination and sumoylation are driven by phosphorylation at specific sites (3), that prolyl isomerization is required for sumoylation of topoisomerases in drug-stabilized DNA cleavage complexes. Specific Aim IV will test the hypotheses that (1) repair enzymes known from lower phyla, but not yet discovered in humans, are present at sites of topoisomerase mediated DNA damage (2) that different sets of repair enzymes are recruited to replication forks damaged by topo I poisons, topo II poisons and hydroxyurea as a result of differences in fork damage caused by these agents. AIM IV下的研究还有望产生有关修复酶募集到损坏的叉子的序列和动力学的新信息，以及有关修复蛋白的直接相互作用和叉子修复步骤的直接相互作用的信息。