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是由凝聚素复合体介导的，它在多种生物学过程中也起着关键作用。 2条途径，包括DNA复制重新启动和DNA双链断裂（DSB）修复下的遗传毒性应激。 3在脊椎动物中，核心粘附素复合物由SMC 1，SMC 3，RAD 21， 4和第四亚基SA 1或SA 2。核心粘附素亚基的种系突变导致广泛的 5种人类疾病统称为“粘连蛋白病”，并增加癌症的发病率。我们目前 6.对粘附素功能的理解是基于粘附素DNA结合仅被介导的模型 7通过粘附素环对dsDNA的非特异性截留。然而，这些模型无法解释 许多研究的8个观察结果表明，在特定的 9结构和序列沿着基因组。我们最近发表了一项新的观察， 10项研究证明SA 1特异性结合双链端粒DNA。此外，我们发现， 11 SA 2显示出对ssDNA缺口的高结合特异性。这些新结果提出了一些根本性的问题 1/2关于SA 1/2-DNA复合物的结构和动力学及其在保持基因组完整性中的作用。 13我们假设，DNA序列和结构依赖性DNA结合的粘附素SA 1/2保存 14遗传毒性胁迫下的基因组完整性。拟议的工作测试这一假设在以下几个方面。 15首先，使用批量分析和单分子原子力和实时荧光显微镜成像 16的蛋白质的DNA，我们将确定域SA 1/2介导的DNA结合，和影响 17个核小体的DNA结合动力学。第二，独特的粘蛋白环的机制 18独立的SA 1介导的姐妹端粒的凝聚过程仍然是未知的。我们将测试“多站点SA 1- 19 TRF 1-TIN 2介导的DNA-DNA桥接”姐妹端粒凝聚模型。我们将定义 20端粒粘附素复合物，通过使用一种新的静电力显微镜（EFM）成像技术， 21揭示了组装蛋白质结构中的DNA路径。研究蛋白质募集的顺序步骤 22和由SA 1-TRF 1-TIN 2蛋白复合物介导的DNA-DNA桥接，我们将使用实时荧光 图23限制在微流体装置中的纳米通道中的DNA分子的显微镜成像。最后，为了确定 SA 1/2在防止DNA损伤诱导的基因组和端粒不稳定性中的作用，我们将研究SA 1/2在预防DNA损伤诱导的基因组和端粒不稳定性中的作用。 图25 SA 1/2 DNA结合突变体和蛋白质消耗对DNA末端连接效率的影响 26培养细胞中的DNA DSB修复。此外，我们假设SA 1介导的端粒凝聚阻止了 27 DNA损伤诱导的端粒不稳定性。为了探索这一假设，我们将研究SA 1 DNA结合 细胞暴露于遗传毒性物质后，28种突变体和蛋白质缺失影响端粒完整性， 29个损伤端粒。这些结果将极大地推进我们对不同细胞中粘附素功能的理解。 30个基因组维持途径。 31 32 33 34 35 36 37 38 39 40 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