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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

联系人 PD/PI：王红 1 姐妹染色单体凝聚力由黏连蛋白复合物介导，该复合物在多种生物中也发挥着关键作用 2 种途径包括 DNA 复制重启和基因毒性应激下的 DNA 双链断裂 (DSB) 修复。 3 在脊椎动物中，核心粘连蛋白复合物由由 SMC1、SMC3、RAD21、 4和第四子单元SA1或SA2。核心粘连蛋白亚基的种系突变导致广泛的 5 种人类疾病统称为“粘连病”，并导致癌症发病率增加。我们目前 6 对粘连蛋白功能的理解基于仅介导粘连蛋白 DNA 结合的模型 7 通过粘连蛋白环非特异性捕获 dsDNA。然而这些模型并不能解释大量研究中的 8 个观察结果证明了粘连蛋白复合物在特定位置的定位沿基因组的 9 个结构和序列。我们最近发表了一项新的单分子观察结果 10 项研究表明 SA1 与双链端粒 DNA 特异性结合。此外，我们发现 11 SA2 对 ssDNA 缺口表现出高结合特异性。这些新结果提出了一些基本问题 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 我们假设粘连蛋白 SA1/2 保留 DNA 序列和结构依赖性 DNA 结合 14 基因毒性胁迫下的基因组完整性。拟议的工作通过以下方式检验这一假设。 15 首先，使用批量分析以及单分子原子力和实时荧光显微镜成像 DNA 上的 16 种蛋白质，我们将识别 SA1/2 上介导 DNA 结合的结构域，以及 17 核小体的 DNA 结合动力学。二、独特的粘连环的机制 18个独立的SA1介导的姐妹端粒凝聚过程仍不清楚。我们将测试“多站点SA1- 19 TRF1-TIN2 介导的 DNA-DNA 桥接”姐妹端粒凝聚力模型。我们将定义以下结构： 20 通过使用新型静电力显微镜 (EFM) 成像技术来观察端粒粘连蛋白复合物图 21 揭示了组装蛋白质结构内的 DNA 路径。研究蛋白质招募的连续步骤 22 和 SA1-TRF1-TIN2 蛋白复合物介导的 DNA-DNA 桥接，我们将使用实时荧光 23 限制在微流体装置纳米通道内的 DNA 分子的显微镜成像。最后，确定 SA1/2 在预防 DNA 损伤引起的基因组和端粒不稳定方面的 24 种作用，我们将研究 25 SA1/2 DNA 结合突变体和蛋白质消耗对连接远端 DNA 末端效率的影响 26 培养细胞中的 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。为了探索这个假设，我们将研究 SA1 DNA 结合如何细胞暴露于基因毒性剂后，28 个突变体和蛋白质消耗会影响端粒完整性 29 损害端粒。这些结果将极大地增进我们对多种粘连蛋白功能的理解 30 条基因组维护途径。 31 32 33 34 35 36 37 38 39 40 项目总结/摘要第 7 页

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

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA.

DOI：
10.1093/nar/gkab1142
发表时间：
2021-12-16
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Kaur P;Barnes R;Pan H;Detwiler AC;Liu M;Mahn C;Hall J;Messenger Z;You C;Piehler J;Smart RC;Riehn R;Opresko PL;Wang H
通讯作者：
Wang H

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

端粒DNA上TRF1-TIN2介导的反式相互作用的结构，动力学和调节。

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