Genomic Stability and RecQ DNA Helicases in Yeast

描述(由申请人提供)：基因组完整性对人类健康和所有物种的生存至关重要。基因组不稳定性的一个主要来源是同源重组(HR)的刺激，当复制叉在DNA模板中的损伤处停止时就会发生。尽管细胞周期检查点和DNA修复酶通常高保真地修复此类损伤，但这些过程中的缺陷与人类疾病和癌症有关。其中一种名为Bloom综合征的疾病是由BLM缺陷引起的，BLM是RecQ DNA解旋酶家族的成员之一。BLM与DNA拓扑异构酶III和一个新发现的亚基Rmi1一起作用，抑制姐妹染色单体交换。这种复合体的基本性质在低等真核生物(如发芽酵母)中的保守性得到了强调。与人类一样，酵母中同源的SGS1-top3-Rmi1(STR)复合体的缺失会导致基因组不稳定，并增强对DNA损伤的敏感性。在这个项目中，我们将利用酵母的生物化学和遗传学来确定带有相扑的蛋白质的翻译后修饰如何调节细胞在没有STR的情况下使用的DNA修复途径。在目标1中，我们将确定SLX5-SLX8 Ub连接酶的生化和遗传功能，该连接酶是在没有STR的情况下生存所必需的。我们将检验这一假设，即SLX5-SLX8活性导致蛋白酶体的多和甲基化蛋白的破坏。体内和体外试验将被用来确定SLX5-SLX8泛素化的SUMoylated蛋白质如何抑制基因组的不稳定性。这将涉及识别相关的体内靶蛋白，以及表征酶的首选底物，可能是一种特定形式的多相扑。在目标2中，我们将研究Wss1的功能，它是一个新的参与者，在控制苏莫化和基因组稳定性。我们将使用体外试验和各种相扑底物来确定Wss1是否是相扑异肽酶。在目标3中，我们将确定在某些DNA修复突变体中出现的多相扑结合物的更广泛的意义。当这些突变体不能聚合相扑链时，我们将检查它们的表型，并鉴定其他DNA修复突变体，这些突变体可以引起多相扑蛋白。我们还将通过确定ULP2的突变等位基因如何抑制sgs1slx5突变的致死性来研究Ulp2异肽酶在基因组维护中的作用。 公共卫生相关性：基因组完整性对于包括人类在内的所有生物体的健康和生存能力至关重要。例如，布卢姆综合征(BS)患者缺乏BLM蛋白，并遭受基因组不稳定的痛苦，最终导致癌症。这个项目试图用酵母作为一个模型系统来表征在没有BLM的情况下运作的DNA修复途径。该项目将利用这一模型系统的众所周知的特征来确定相扑对蛋白质修饰的作用，并表征在没有这种修饰的情况下发挥作用的替代修复途径。因此，这项研究将对维持正常和BS细胞基因组稳定的因素和遗传途径提供新的理解。

DESCRIPTION (provided by applicant): Genome integrity is essential for human health and the viability of all species. A major source of genome instability is the stimulation of homologous recombination (HR) that occurs when replication forks arrest at lesions in the DNA template. Although cell-cycle checkpoints and DNA repair enzymes typically repair such lesions with high fidelity, defects in these processes are associated with human disease and cancer. One such disease, Bloom Syndrome, arises from defects in BLM, a member of RecQ family of DNA helicases. BLM acts together with DNA topoisomerase III and a newly-identified subunit, Rmi1, to suppress sister chromatid exchange. The fundamental nature of this complex is underscored by its conservation in lower eukaryotes such as budding yeast. As in humans, loss of the homologous Sgs1-Top3-Rmi1 (STR) complex in yeast results in genome instability and enhanced sensitivity to DNA damage. In this project we will exploit the biochemistry and genetics of yeast to determine how post-translational modification of proteins with SUMO regulates the DNA repair pathways that the cell uses in the absence of STR. In Aim 1 we will determine the biochemical and genetic function of the Slx5-Slx8 Ub ligase which is essential for viability in the absence of STR. We will test the hypothesis that Slx5-Slx8 activity leads to the proteasomal destruction of poly- sumoylated proteins. In-vivo and in-vitro assays will be used to determine how the ubiquitination of sumoylated proteins by Slx5-Slx8 suppresses genome instability. This will involve identifying relevant in-vivo target proteins as well as characterizing the enzyme's preferred substrate which may be a specific form of poly-SUMO chains. In Aim 2 we will examine the function of Wss1 which is a new player in the control of sumoylation and genome stability. We will determine whether Wss1 is a SUMO isopeptidase using in-vitro assays and a variety of sumoylated test substrates. In Aim 3 we will determine the broader significance of poly- SUMO conjugates that arise in certain DNA repair mutants. We will examine the phenotype of such mutants when they are unable to polymerize SUMO chains, and identify additional DNA repair mutants that give rise to poly-sumoylated proteins. We will also examine the role of the Ulp2 isopeptidase in genome maintenance by determining how a mutant allele of ULP2 suppresses the lethality of sgs1 slx5 mutants. PUBLIC HEALTH RELEVANCE: Genome integrity is essential for the health and viability of all organisms, including humans. For example, patients with Bloom Syndrome (BS) lack the BLM protein and suffer from genome instability that eventually leads to cancer. This project seeks to characterize the DNA repair pathways that operate in the absence of BLM using yeast as a model system. The project will exploit well-known features of this model system to determine role of protein modification by SUMO and to characterize alternative repair pathways that function in the absence of this modification. Thus, this research will provide new understanding about the factors and genetic pathways that maintain genome stability in normal and BS cells.