Genomic Stability and RecQ DNA Helicases in Yeast

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家族基因的突变已被证明可导致三种不同的遗传疾病[Bloom综合征（BLM）， Werner综合征（WRN）和rothmond - thomson综合征（RTS）的一个子集]。来自这些患者的细胞显示出过多的DNA重排，这表明它们的主要缺陷是未能保持基因组的稳定性。出芽酵母中唯一的RecQ同源物Sgs1存在于与DNA拓扑异构酶III （Top3）的复合体中，并作为人类BLM的模型，该复合体也与stop3结合。我们利用酵母系统来鉴定与RecQ-Top3复合物功能冗余的遗传途径。其中两种途径由Slx1-Slx4和SIx5-SIx8蛋白复合物定义。我们已经证明Slx1-SIx4是一个5'-flap内切酶，而SIx5-Slx8没有已知的活性。本研究拟通过对Slxl-4、Slx5-8和Sgsl-Top3这三个复合物的生化和遗传分析，探讨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和表征Slx1-SIx4蛋白复合物。新的亚基将被鉴定并测试它们对5'-皮瓣内切酶活性的影响。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。基因组稳定性将通过测量重组频率和研究Slx1-4复合物在控制rDNA重复结构中的作用来测定突变细胞。蛋白质的核定位也将被确定。在Aim 2中，我们将类似地纯化和表征Slx5-SIx8复合物。Slx5和Slx8亚基是无名指蛋白，表明Slx5-8复合物可能参与蛋白修饰。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。一个积极的结果将通过搜索特定的底物修饰扩展。在Aim 3中，Sgsl-Top3将被纯化，并使用我们的遗传分析建议的底物检测DNA解旋酶和拓扑异构酶活性。

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.