Mechanism of spontaneous and DSB-induced repair

自发修复和 DSB 诱导修复的机制

• 批准号: 6654394
• 负责人: Rodney J. ROTHSTEIN
• 金额: $ 58万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6654394
• 关键词: DNA binding protein; DNA damage; DNA repair; DNA topoisomerases; alleles; cell cycle; fungal genetics; fungal proteins; gene interaction; genetic recombination; green fluorescent proteins; helicase; hydroxyurea; intermolecular interaction; molecular genetics; phosphorylation; posttranslational modifications; protein degradation; protein localization; protein purification; protein structure function; yeasts

Our goal is to understand the mechanism underlying the recognition and repair of DNA damage. We will use yeast as outlined in the specific aims: (1) The Rad52 DNA repair protein: We will continue our molecular genetic characterization of the Rad52 DNA repair pathway using newly developed Rad52-GFP fusions. We will continue to explore Rad52 functional domains and analyze the pathways defined by novel gamma-ray sensitive, hyper- recombination rad52 alleles. We will also explore the post translational modification that produces multiple Rad52 protein species. (2) Sml1, a negative regulator of RNF: We will investigate the regulatory circuitry of the Rnr inhibitor, Sml1. We will determine which pathways and what modifications regulate Sml1 degradation during S phase and after DNA damage. We will investigate the role of other proteins in Sml1 phosphorylation and/or regulation and develop a new tool to allow the visualization of the DNA damage response in living cells. (3) The Top3/Sgs1 DNA topoisomerase/helicase complex: We will continue to explore the genetic and biochemical interactions between Top3 and Sgs1. We will isolate and identify new alleles of SGS1 that separate top3 suppression from its DNA damage sensitivity. To further define the TOP3 function, we will attempt to isolate non-sgs1 suppressors of top3 as well as suppressors of catalytically inactive Top3. We will investigate those Top3 functions that do not require Sgs1. We will further characterize SHU1, a suppressor of sgs 1 hydroxyurea sensitivity. We will also explore the cell biology and biochemistry of this important protein complex. These combined genetic, biochemical and cell biological approaches to the many issues related to the recognition and repair of DNA damage in yeast will continue to yield new insights into this important biological process.

我们的目标是了解DNA损伤识别和修复的基础机制。 我们将使用特定目的中概述的酵母：（1）RAD52 DNA修复蛋白：我们将使用新开发的RAD52-GFP融合来继续我们的Rad52 DNA修复途径的分子遗传表征。 我们将继续探索RAD52功能域，并分析由新型γ-射线敏感，超重组RAD52等位基因定义的途径。 我们还将探索产生多种RAD52蛋白质物种的翻译后修饰。 （2）SML1，RNF的负调节剂：我们将研究RNR抑制剂SML1的调节电路。 我们将确定哪些途径和哪些修饰调节S相和DNA损伤后SML1降解。 我们将研究其他蛋白质在SML1磷酸化和/或调节中的作用，并开发一种新工具，以允许在活细胞中可视化DNA损伤反应。 （3）TOP3/SGS1 DNA拓扑异构酶/解旋酶复合物：我们将继续探索TOP3和SGS1之间的遗传和生化相互作用。 我们将隔离并确定SGS1的新等位基因，该等位基因将TOP3抑制与其DNA损伤敏感性分开。 为了进一步定义TOP3函数，我们将尝试分离TOP3的非SGS1抑制剂以及催化无效TOP3的抑制剂。我们将研究不需要SGS1的TOP3函数。 我们将进一步表征SHU1，Shu1是SGS 1羟基脲敏感性的抑制剂。 我们还将探索这种重要的蛋白质复合物的细胞生物学和生物化学。这些结合遗传，生化和细胞生物学方法对于与酵母中DNA损伤的识别和修复有关的许多问题将继续对这一重要的生物学过程产生新的见解。