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。基因组不稳定性的一个主要来源是当复制叉在DNA模板中的损伤处停止时发生的同源重组（HR）的刺激。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。一种这样的疾病，布卢姆综合征，由BLM（DNA解旋酶的RecQ家族的成员）中的缺陷引起。BLM与DNA拓扑异构酶III和一个新发现的亚基Rmi 1一起抑制姐妹染色单体交换。这种复合物的基本性质是强调其在低等真核生物如芽殖酵母中的保守性。与人类一样，酵母中同源Sgs 1-Top3-Rmi 1（STR）复合物的丢失导致基因组不稳定性和对DNA损伤的敏感性增强。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蛋白质的SUMO翻译后修饰如何调节细胞在STR缺失的情况下使用的DNA修复途径。在目的1中，我们将确定Slx 5-Slx 8 Ub连接酶的生物化学和遗传功能，该连接酶在STR缺失的情况下对生存力至关重要。我们将测试Slx 5-Slx 8 Ub连接酶的假设。Slx 8活性导致多sumoylated蛋白质的蛋白酶体破坏。将使用体内和体外测定来确定Slx 5-Slx 8对sumoylated蛋白的泛素化如何抑制基因组不稳定性。这将涉及鉴定相关的体内靶蛋白以及表征酶的优选底物，其可能是聚SUMO链的特定形式。在目标2中，我们将研究Wss 1的功能，Wss 1是控制类小泛素化和基因组稳定性的新参与者。我们将确定Wss 1是否是一个SUMO异肽酶使用体外试验和各种sumoylated测试基板。在目标3中，我们将确定在某些DNA修复突变体中出现的多聚SUMO缀合物的更广泛的意义。我们将研究这些突变体的表型时，他们不能SUMO链，并确定额外的DNA修复突变体，产生聚sumoylated蛋白。我们还将研究的Ulp 2异肽酶在基因组维护中的作用，通过确定如何突变的等位基因ULP 2抑制sgs 1 slx 5突变体的致死性。 公共卫生相关性：基因组完整性对所有生物体（包括人类）的健康和生存能力至关重要。例如，Bloom综合征（BS）患者缺乏BLM蛋白，并患有最终导致癌症的基因组不稳定性。该项目旨在描述在没有BLM的情况下使用酵母作为模型系统的DNA修复途径。该项目将利用该模型系统的众所周知的功能来确定SUMO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，这项研究将提供新的理解的因素和遗传途径，维持基因组稳定性在正常和BS细胞。

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.