Genomic Stability and RecQ DNA Helicases in Yeast

酵母中的基因组稳定性和 RecQ DNA 解旋酶

• 批准号: 7035787
• 负责人: STEVEN J. BRILL
• 金额: $ 29.61万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035787
• 关键词: DNA damage; DNA repair; DNA topoisomerases; Saccharomyces cerevisiae; cell cycle; endonuclease; enzyme activity; fungal genetics; fungal proteins; gene mutation; gene rearrangement; genetic recombination; helicase; nucleic acid repetitive sequence; protein localization; protein structure function; recombinant proteins; ubiquitin

DESCRIPTION (provided by applicant): Loss of genome stability is associated with a number of human diseases that predispose patients to cancer. In particular, mutations in the genes encoding the RecQ family of DNA helicases have been shown to cause three distinct genetic diseases [Bloom syndrome (BLM), Werner syndrome (WRN), and a subset of Rothmund-Thomson syndrome (RTS)]. Cells from these patients display excessive DNA rearrangements suggesting that their primary defect is the failure to maintain genome stability. The only RecQ homolog in budding yeast, Sgs1, exists in a complex with DNA topoisomerase III (Top3) and serves as a model for human BLM which also bindsTop3. We have exploited the yeast system to identify genetic pathways that are functionally redundant with the RecQ-Top3 complex. Two of these pathways are defined by the Slx1-Slx4 and SIx5-SIx8 protein complexes. We have shown that Slx1-SIx4 is a 5'-flap endonuclease while SIx5-Slx8 has no known activity. Here we propose to explore the mechanism by which the RecQ-Top3 complex controls genome stability using biochemical and genetic analysis of these three complexes: Slxl-4, Slx5-8, and Sgsl-Top3. In Aim1 we will purify and characterize the Slx1-SIx4 protein complex from yeast. New subunits will be identified and tested for their effect on 5'-flap endonuclease activity. A structure-function analysis of both subunits will be conducted and the role of these proteins in cell-cycle checkpoint control will be tested. Genome stability will be assayed in mutant cells by measuring recombination frequencies and by investigating the role of the Slx1-4 complex in controlling rDNA repeat structure. The proteins' nuclear localization will also be determined. In Aim 2 we will similarly purify and characterize the Slx5-SIx8 complex. The Slx5 and Slx8 subunits are RING-finger proteins suggesting that the Slx5-8 complex may be involved in protein modification. Using purified recombinant protein we will test Slx5-8 for E3 ligase activity using Smt3 or ubiquitin as ligand. A positive result will be extended by searching for specific substrates of the modification. In Aim 3 Sgsl-Top3 will be purified and tested for DNA helicase and topoisomerase activity using substrates that have been suggested by our genetic analysis.

描述（由申请人提供）：基因组稳定性的丧失与许多使患者易患癌症的人类疾病有关。 特别是，编码 DNA 解旋酶 RecQ 家族的基因突变已被证明会导致三种不同的遗传疾病 [布卢姆综合征 (BLM)、沃纳综合征 (WRN) 和罗斯蒙德-汤姆森综合征 (RTS) 的一个子集]。这些患者的细胞表现出过度的 DNA 重排，表明他们的主要缺陷是无法维持基因组稳定性。芽殖酵母中唯一的 RecQ 同源物 Sgs1 存在于与 DNA 拓扑异构酶 III (Top3) 的复合物中，并作为也结合 Top3 的人类 BLM 的模型。 我们利用酵母系统来识别与 RecQ-Top3 复合物功能冗余的遗传途径。 其中两条途径由 Slx1-Slx4 和 SIx5-SIx8 蛋白复合物定义。 我们已经证明 Slx1-SIx4 是一种 5'-flap 核酸内切酶，而 SIx5-Slx8 没有已知的活性。在这里，我们建议利用这三个复合物（Slxl-4、Slx5-8 和 Sgsl-Top3）的生化和遗传分析来探索 RecQ-Top3 复合物控制基因组稳定性的机制。 在 Aim1 中，我们将从酵母中纯化并表征 Slx1-SIx4 蛋白质复合物。 新的亚基将被鉴定并测试其对 5'-flap 核酸内切酶活性的影响。 将进行两个亚基的结构功能分析，并测试这些蛋白质在细胞周期检查点控制中的作用。 通过测量重组频率和研究 Slx1-4 复合物在控制 rDNA 重复结构中的作用，将在突变细胞中测定基因组稳定性。 蛋白质的核定位也将被确定。 在目标 2 中，我们将类似地纯化和表征 Slx5-SIx8 复合物。 Slx5 和 Slx8 亚基是环指蛋白，表明 Slx5-8 复合物可能参与蛋白质修饰。 使用纯化的重组蛋白，我们将使用 Smt3 或泛素作为配体测试 Slx5-8 的 E3 连接酶活性。 通过寻找修饰的特定底物将扩展积极的结果。 在目标 3 中，将使用我们的遗传分析建议的底物纯化 Sgsl-Top3 并测试 DNA 解旋酶和拓扑异构酶活性。