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相关。 特别地，编码RecQ家族DNA解旋酶的基因中的突变已显示引起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-汤姆森综合征（RTS）的子集]。来自这些患者的细胞显示出过度的DNA重排，这表明它们的主要缺陷是未能维持基因组稳定性。芽殖酵母中唯一的RecQ同源物Sgs 1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BLM的模型。 我们已经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Slx 1-Slx 4和SIx 5-SIx 8蛋白复合物定义。 我们已经表明Slx 1-SIx 4是一种5 '-瓣状核酸内切酶，而SIx 5-Slx 8没有已知的活性。在这里，我们提出探索RecQ-Top3复合物控制基因组稳定性的机制，使用这三种复合物的生化和遗传分析：Slxl-4，Slx 5 -8和Sgsl-Top3。 在目标1中，我们将纯化和表征来自酵母的Slx 1-SIx 4蛋白复合物。 将鉴定新的亚基并测试它们对5 '-瓣状核酸内切酶活性的影响。 将对这两种亚基进行结构-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。 将通过测量重组频率和研究Slx 1 -4复合物在控制rDNA重复结构中的作用，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Slx 5-SIx 8复合物。Slx 5和Slx 8亚基是环指蛋白，表明Slx 5 -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Smt 3或泛素作为配体测试Slx 5 -8的E3连接酶活性。 一个积极的结果将通过寻找特定的基板的修改。 在目的3中，将纯化Sgsl-Top3并使用我们的遗传分析所建议的底物测试DNA解旋酶和拓扑异构酶活性。