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: 7218002
• 负责人: STEVEN J. BRILL
• 金额: $ 28.75万
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7218002
• 关键词: Biochemical Genetics; Biological Assay; Bloom Syndrome; Cell Cycle Checkpoint; Cells; Complex; Defect; Failure; Family; Flap Endonucleases; Frequencies; Gene Rearrangement; Genes; Genetic; Genetic Recombination; Genome Stability; Hereditary Disease; Homologous Gene; Human; Ligands; Malignant Neoplasms; Measures; Modeling; Modification; Mutation; Nuclear Protein; Nuclear Proteins; Numbers; Pathway interactions; Patients; Post-Translational Protein Processing; Proteins; Recombinant DNA; Recombinant Proteins; Ribosomal DNA; Role; Rothmund-Thomson syndrome; Saccharomycetales; Structure; System; Testing; Topoisomerase; Topoisomerase III; Ubiquitin; Werner Syndrome; Yeast Protein Complex; Yeasts; genetic analysis; helicase; human RBX1 protein; human disease; mutant; ubiquitin-protein ligase

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人类疾病有关，这些疾病使患者容易患上癌症。特别是，编码DNA解旋酶RecQ家族的基因突变已被证明导致三种不同的遗传病[Bloom综合征(BLM)、Werner综合征(WRN)和Rothmund-Thomson综合征(RTS)的子集]。这些患者的细胞表现出过度的DNA重排，这表明他们的主要缺陷是未能维持基因组的稳定。萌芽酵母中唯一的RecQ同源物SGS1存在于DNA拓扑异构酶III(Top3)的复合体中，并作为人类BLM的模型，该模型也与Top3结合。我们已经利用酵母系统来识别与RecQ-top3复合体在功能上多余的遗传途径。其中两条通路由SLX1-SLX4和SIx5-SIx8蛋白复合体定义。我们发现SIx1-SIx4是一种5‘-翻盖核酸内切酶，而SIx5-SIx8没有已知的活性。在这里，我们建议通过对三个复合体：slx1-4、slx5-8和sgsl-top3的生化和遗传分析来探索RecQ-top3复合体控制基因组稳定性的机制。在Aim1中，我们将从酵母中纯化并鉴定SLX1-SIx4蛋白复合体。新的亚基将被识别并测试它们对5‘-Flat核酸内切酶活性的影响。将对这两个亚基进行结构-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复合体在控制rDNA重复结构中的作用，将在突变细胞中测试基因组的稳定性。蛋白质的核定位也将得到确定。在目标2中，我们将类似地纯化和表征SLX5-SIx8复合体。SLX5和SLX8亚基是环指蛋白，提示SLX5-8复合体可能参与蛋白质修饰。使用纯化的重组蛋白，我们将以Smt3或泛素为配基，检测SLX5-8的E3连接酶活性。通过寻找修饰的特定底物，阳性结果将得到扩展。在AIM中，3SGSL-top3将被提纯，并使用我们的遗传分析所建议的底物来测试DNA解旋酶和拓扑异构酶的活性。