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，用于快速纯化共价连接到 DNA 的蛋白质。 CXLA 产生的蛋白质的数量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一些关于拓扑异构酶翻译后修饰的结构和功能的假设和模型，这些拓扑异构酶是由抗癌药物和治疗靶向而产生的。具体目标我将检验拓扑异构酶 I 的苏酰化参与喜树碱拓扑异构酶 I 介导的 DNA 损伤的检测、信号传导和/或处理的假设。将测试苏酰化特定功能的模型，并在必要时测试替代假设。具体目标 II 将检验拓扑异构酶 II 的泛素家族修饰及其功能随同工酶、其功能以及稳定拓扑异构酶 II-DNA 裂解复合物的抗癌药物而变化的假设。具体目标 III 将测试拓扑异构酶磷酸化功能的几个假设：(1)、DNA 切割需要特定位点的磷酸化 (2)、泛素化和苏酰化是由特定位点的磷酸化驱动的 (3)、脯氨酰异构化是药物稳定的 DNA 裂解复合物中拓扑异构酶的苏酰化所需。具体目标 IV 将测试以下假设：(1) 已知的低等门修复酶，但尚未在人类中发现，存在于拓扑异构酶介导的 DNA 损伤位点 (2) 不同组的修复酶被招募到由拓扑异构酶介导的 DNA 损伤损伤的复制叉上。拓扑异构酶 I 毒物、拓扑异构酶 II 毒物和羟基脲，由于这些药剂造成的叉损伤不同。 Aim IV 下的研究预计还将产生有关修复酶招募到受损叉的序列和动力学的新信息，以及有关修复蛋白直接相互作用和叉修复步骤的信息。