Sequence and Structure Specific DNA Binding by Cohesin and Genome Stability

粘连蛋白的序列和结构特异性 DNA 结合以及基因组稳定性

基本信息

批准号：
10175465
负责人：
Hong Wang
金额：
$ 11.4万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10175465
关键词：
3-Dimensional Antigens Architecture Atomic Force Microscopy Binding Biological Biological Assay Biological Process CSPG6 gene Cell Survival Chromatin Chromosomes Complex Cultured Cells DNA DNA Binding DNA Damage DNA Double Strand Break DNA Sequence DNA Structure DNA biosynthesis DNA replication fork Diffusion Disease Distal Double Strand Break Repair Electrostatics Exposure to Fluorescence Fluorescence Microscopy Genome Genome Stability Genomic Instability Genomics Genotoxic Stress Germ-Line Mutation Goals Imaging Techniques Incidence Lead Link Maintenance Malignant Neoplasms Mediating Microfluidic Microchips Modeling Movement Mutagens Nucleoproteins Nucleosomes Pathway interactions Play Process Property Proteins Publishing RNA Regulator Genes Reporting Role Sister Sister Chromatid Site Specificity Structure Surgical Flaps TERF1 gene TINF2 gene Telomeric Repeat Binding Protein 1 Tertiary Protein Structure Testing Time Vertebrates Work base cancer cell cancer therapy chemotherapy cohesin cohesion ds-DNA genome integrity human disease imaging platform innovation insight man microscopic imaging mutant nanochannel novel preservation prevent protein complex protein structure recruit single molecule telomere trafficking

项目摘要

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

联系PD/PI：Wang，Hong 1个姐妹染色质凝聚是由粘蛋白复合物介导的，粘蛋白复合物也在潜水员生物学中起关键作用在遗传毒性应激下的2条途径（包括DNA复制重新启动和DNA双链断裂（DSB）修复）。 3在脊椎动物中，核心粘着素复合物由一个由SMC1，SMC3，Rad21， 4和第四个亚基SA1或SA2。核心粘着素亚基中的种系突变导致广泛 5种人类疾病统称为“粘毒素病，并增加了癌症的事件。我们的当前 6对粘蛋白功能的理解是基于模型，在该模型中，粘蛋白DNA结合仅介导 7通过粘蛋白环对DsDNA的非特异性捕集。但是，这些模型无法解释 8来自众多研究的观察结果，证明了粘蛋白复合物在特定沿基因组的9个结构和序列。我们最近发表了一种来自单分子的新颖观察结果 10个研究表明，SA1专门与双链远程远程DNA结合。此外，我们发现 11 SA2显示ssDNA间隙的高结合规格。这些新结果提出了基本问题 12关于SA1/2-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我们假设通过粘蛋白SA1/2保留DNA序列和结构依赖性DNA结合 14基因组完整性在遗传毒性应激下。提出的工作通过以下方式检验了这一假设。 15首先，使用散装测定和单分子原子力和实时荧光显微镜成像 DNA上的16个蛋白质，我们将在SA1/2上识别介导DNA结合的域，以及其DNA结合动力学上的17个核小组。其次，独特的粘蛋白环的基础机制 18独立的SA1介导的姐妹端粒凝聚过程仍然未知。我们将测试“多站点SA1- 19 trf1-Tin2介导的DNA-DNA桥接“姐妹端粒凝聚力的模型”。我们将定义 20通过使用新型的静电力显微镜（EFM）成像技术到远程仪表符络合物复合物 21揭示组装蛋白结构内的DNA路径。研究蛋白质募集的顺序步骤 22和DNA-DNA桥接由SA1-TRF1-TIN2蛋白复合物介导的，我们将使用实时荧光 23限于微流体设备中纳米通道的DNA分子的显微镜成像。最后，确定 SA1/2的24个在防止DNA损伤引起的基因组和远程静态不稳定性中的作用，我们将研究 25 SA1/2 DNA结合突变体和蛋白质耗竭对连接远端DNA末端效率的影响培养细胞中的26 DNA DSB修复。此外，我们假设SA1介导的端粒凝聚力可防止 27 DNA损伤引起的端粒不稳定。为了探索这一假设，我们将研究SA1 DNA如何结合 28突变体和蛋白质部署会影响细胞暴露于遗传毒性剂后的端粒完整性 29损伤端粒。这些结果将大大提高我们对潜水员粘素功能的理解 30个基因组维护途径。 31 32 33 34 35 36 37 38 39 40 项目摘要/摘要页面7

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

PARP1 associates with R-loops to promote their resolution and genome stability.

DOI：
10.1093/nar/gkad066
发表时间：
2023-03-21
期刊：
Nucleic acids research
影响因子：
14.9
作者：
通讯作者：

Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein.

DOI：
10.1093/nar/gky875
发表时间：
2018-11-30
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Kaur P;Longley MJ;Pan H;Wang H;Copeland WC
通讯作者：
Copeland WC

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.

DOI：
10.1093/nar/gkac1089
发表时间：
2022-11-11
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
Li, Zhuo;Kaur, Parminder;Lo, Chen-Yu;Chopra, Neil;Smith, Jamie;Wang, Hong;Gao, Yang
通讯作者：
Gao, Yang

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

DOI：
10.1016/j.jbc.2021.101080
发表时间：
2021-09
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Pan H;Kaur P;Barnes R;Detwiler AC;Sanford SL;Liu M;Xu P;Mahn C;Tang Q;Hao P;Bhattaram D;You C;Gu X;Lu W;Piehler J;Xu G;Weninger K;Riehn R;Opresko PL;Wang H
通讯作者：
Wang H

Measuring UV Photoproduct Repair in Isolated Telomeres and Bulk Genomic DNA.

测量分离端粒和大量基因组 DNA 中的紫外光产物修复。