Sequence and Structure Specific DNA Binding by Cohesin and Genome Stability

联系PD/PI：Wang，Hong 1姐妹染色单体的凝聚力是由粘附素复合体介导的，粘附素复合体在多种生物学中也起着关键作用 基因毒性应激下DNA复制重启和DNA双链断裂修复两条途径。 3在脊椎动物中，核心粘附素复合体由SMC1、SMC3、RAD21、 4和第四亚单位Sa1或Sa2。核心粘附素亚基的胚系突变导致广泛的 人类5种疾病统称为“粘连病症”，并增加癌症的发病率。我们目前的情况 6对粘附素功能的理解是基于粘附素DNA结合被排他地介导的模型 7通过粘附素环非特异性地捕获dsDNA。然而，这些模型无法解释 8从众多研究中观察到的粘附素复合体在特定区域的定位 9基因组上的结构和序列。我们最近发表了一项来自单分子的新观察 10项研究表明SA1与双链端粒DNA特异结合。另外，我们发现， 11 SA2对单链DNA间隙具有很高的结合特异性。这些新的结果提出了根本的问题 12关于SA1/2-DNA复合体的结构和动力学及其在保持基因组完整性方面的作用。 13我们假设通过粘连蛋白SA1/2结合的DNA序列和结构依赖于DNA 14基因毒性胁迫下的基因组完整性。这项拟议的工作通过以下方式检验了这一假说。 15首先，使用整体分析和单分子原子力和实时荧光显微镜成像 16个蛋白质在DNA上，我们将确定SA1/2上介导DNA结合的结构域，以及 17个核小体的DNA结合动力学。第二，独特的粘附素环背后的机制 18独立的SA1介导的姐妹端粒凝聚过程尚不清楚。我们将测试“多站点SA1-- 19 TRF1-TIN2介导的姊妹端粒凝聚的DNA-DNA桥联模型。我们将定义 20通过使用新的静电力显微镜(EFM)成像技术来检测端粒粘附素复合体 21揭示了组装的蛋白质结构中的DNA路径。研究蛋白质募集的顺序步骤 22和SA1-TRF1-TIN2蛋白复合体介导的DNA-DNA桥联，我们将使用实时荧光 23微流控装置中限制在纳米通道内的DNA分子的显微成像。最后，要确定 24 SA1/2在防止DNA损伤诱导的基因组和端粒不稳定中的作用，我们将研究 25 SA1/2 DNA结合突变体和蛋白质缺失对DNA末端连接效率的影响 26培养细胞的DNA DSB修复。此外，我们假设SA1介导的端粒凝聚力阻止 27 DNA损伤导致端粒不稳定。为了探索这一假设，我们将研究SA1 DNA结合 28个突变和蛋白质耗尽影响细胞暴露于基因毒性药物后的端粒完整性 29例端粒受损。这些结果将极大地促进我们对不同种类的粘附素功能的理解。 30条基因组维护途径。 31 32 33 34 35 36 37 38 39 40岁 项目摘要/摘要第7页

Contact PD/PI: Wang, Hong 1 Sister chromatid cohesion is mediated by the cohesin complex, which also plays key roles in diverse biological 2 pathways including DNA replication restart, and DNA double-strand break (DSB) repair under genotoxic stress. 3 In vertebrates, the core cohesin complex consists of a tripartite ring assembled from SMC1, SMC3, RAD21, 4 and the fourth subunit SA1 or SA2. Germline mutations in core cohesin subunits lead to a wide spectrum of 5 human diseases collectively called “cohesinopathies, and to increased incidence of cancers. Our current 6 understanding of cohesin functions is based on models in which cohesin DNA binding is mediated exclusively 7 through nonspecific entrapment of dsDNA by the cohesin ring. However, these models cannot explain 8 observations from numerous studies demonstrating the localization of the cohesin complex at specific 9 structures and sequences along the genome. We recently published a novel observation from single-molecule 10 studies demonstrating that SA1 binds specifically to double-stranded telomeric DNA. In addition, we found that 11 SA2 displays high binding specificities for ssDNA gaps. These new results raise fundamental questions 12 regarding the structure and dynamics of SA1/2-DNA complexes and their roles in preserving genome integrity. 13 We hypothesize that DNA sequence and structure-dependent DNA binding by cohesin SA1/2 preserve 14 genome integrity under genotoxic stress. The proposed work tests this hypothesis in the following ways. 15 First, using both bulk assays and single-molecule atomic force and real-time fluorescence microscopy imaging 16 of proteins on DNA, we will identify the domains on SA1/2 that mediate DNA binding, and the impact of 17 nucleosomes on their DNA binding dynamics. Second, the mechanism underlying the unique cohesin-ring 18 independent SA1-mediated sister telomere cohesion process is still unknown. We will test the “multi-site SA1- 19 TRF1-TIN2-mediated DNA-DNA bridging” model for sister telomere cohesion. We will define the architecture of 20 the telomeric cohesin complex by using a novel electrostatic force microscopy (EFM) imaging technique to 21 reveal DNA paths within the assembled protein structure. To study the sequential steps in protein recruitment 22 and DNA-DNA bridging mediated by SA1-TRF1-TIN2 protein complexes, we will use real-time fluorescence 23 microscopy imaging of DNA molecules confined to nanochannels in microfluidic devices. Finally, to determine 24 roles for SA1/2 in preventing DNA damage-induced genomic and telomeric instability, we will investigate the 25 impact of SA1/2 DNA binding mutants and protein depletion on the efficiency of joining distal DNA ends during 26 DNA DSB repair in cultured cells. Furthermore, we hypothesize that SA1-mediated telomere cohesion prevents 27 DNA damage-induced telomere instability. To explore this postulate, we will examine how SA1 DNA binding 28 mutants and protein depletion impact telomere integrity after cellular exposure to genotoxic agents that 29 damage telomeres. These results will greatly advance our understanding of the cohesin function in diverse 30 genome maintenance pathways. 31 32 33 34 35 36 37 38 39 40 Project Summary/Abstract Page 7