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能力至关重要。基因组不稳定的一个主要来源是同源重组（HR）的刺激，当复制叉在DNA模板的损伤处停止时发生。虽然细胞周期检查点和DNA修复酶通常以高保真度修复这些病变，但这些过程中的缺陷与人类疾病和癌症有关。其中一种疾病，布卢姆综合征，是由DNA解旋酶RecQ家族成员BLM的缺陷引起的。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强调了它在低级真核生物如芽殖酵母中的保存。与人类一样，酵母中同源Sgs1-Top3-Rmi1 （STR）复合物的缺失导致基因组不稳定和对DNA损伤的敏感性增强。在本项目中，我们将利用酵母的生物化学和遗传学来确定SUMO蛋白的翻译后修饰如何调节细胞在缺乏STR时使用的DNA修复途径。在Aim 1中，我们将确定Slx5-Slx8 Ub连接酶的生物化学和遗传功能，这对于缺乏STR时的生存能力至关重要。我们将验证Slx5-Slx8活性导致多聚合蛋白的蛋白酶体破坏的假设。体内和体外试验将用于确定Slx5-Slx8对sumoylated蛋白的泛素化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，这可能是一种特定形式的聚sumo链。在Aim 2中，我们将研究Wss1的功能，它是控制sumo化和基因组稳定性的新参与者。我们将通过体外实验和多种sumoylated测试底物来确定Wss1是否为SUMO异肽酶。在Aim 3中，我们将确定在某些DNA修复突变体中出现的聚SUMO偶联物的更广泛意义。当这些突变体不能聚合SUMO链时，我们将检查它们的表型，并鉴定产生多聚合蛋白的其他DNA修复突变体。我们还将通过确定Ulp2突变等位基因如何抑制sgs1 slx5突变体的致死率来研究Ulp2异肽酶在基因组维持中的作用。

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.