DNA Recruitment, Regulation & Function of SMC5/6

DNA招募、调控

• 批准号: 323666480
• 负责人: Dr. Markus Räschle
• 金额: --
• 依托单位: Fachgebiet Molekulare Biotechnologie und Systembiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/323666480?language=en
• 关键词: DNA; Recruitment; Regulation; Function; SMC5

Complex regulatory mechanisms control chromatin structure. Hereby protein complexes of the SMC family (cohesin, condensin and SMC5/6) play essential roles. Cohesin and condensin form large rings which embrace DNA duplexes. While cohesin keeps sister chromatids together - from the moment they emerge through DNA replication till the onset of cell division - condensin facilitates chromosome compaction and accurate segregation of one copy of the genetic material to the daughter cells. In contrast, the exact function of SMC5/6 remains unknown. Inactivation of SMC5/6 leads to an immediate cell cycle arrest in mitosis or inaccurate transmission of the genetic material to the daughter cells, respectively. Consistently, compromised SMC5/6 function accelerates tumorigenesis in animal models.Here we propose to study SMC5/6 function in Xenopus egg extracts. Our preliminary results show that SMC5/6 is recruited to plasmid substrates in a replication-dependent fashion. Importantly, we detect plasmid-bound proteins with newly-developed, unbiased proteomic methods. This allows us to accurately quantify all SMC5/6 subunits as well as cohesin and all DNA replication factors.Using this unique system we will address the following questions:1) When and how is SMC5/6 loaded onto DNA?We will monitor protein recruitment to DNA during normal and perturbed DNA replication. We will determine the cellular network regulating the physiological recruitment of SMC5/6 to DNA by identifying the relevant DNA substrates and loading complexes. We will test whether the SMC5/6 proteins form ring-like structures that mediate topological binding of the DNA. We further propose experiments to gain insight into how the distinctive structures of SMC5/6 and its ATPase, sumo and ubiquitin E3 ligase activity relate to its function. 2) What are the functions of SMC5/6?We will generate reagents for the depletion of SMC5/6 or its loading factors from Xenopus egg extracts as well as recombinant proteins for rescue experiments. Using depleted extracts with defined plasmid substrates we will analyze DNA replication and repair intermediates using a variety of established assays. These experiments will directly reveal a possible function of SMC5/6 in the decatenation of replication products or in distinct steps of defined DNA repair pathways.By combining a biochemically tractable system with defined DNA substrates and unbiased, ultra-sensitive proteomic methods, we will for the first time be able to correlate the temporal appearance of DNA intermediates with the assembly of molecular machines involved in the DNA transactions. We anticipate that these methods will provide unprecedented insight into the function and action of the hitherto enigmatic SMC5/6 complex.

复杂的调控机制控制染色质结构。因此，SMC 家族的蛋白质复合物（粘连蛋白、凝缩蛋白和 SMC5/6）发挥着重要作用。粘连蛋白和凝缩蛋白形成包围 DNA 双链体的大环。虽然粘连蛋白将姐妹染色单体保持在一起 - 从它们通过 DNA 复制出现的那一刻起直到细胞分裂开始 - 凝缩蛋白促进染色体压缩以及将遗传物质的一份副本准确分离到子细胞。相比之下，SMC5/6 的确切功能仍不清楚。 SMC5/6 失活分别导致有丝分裂中的细胞周期立即停滞或遗传物质向子细胞的不准确传递。一致地，受损的 SMC5/6 功能会加速动物模型中的肿瘤发生。在此，我们建议研究非洲爪蟾卵提取物中的 SMC5/6 功能。我们的初步结果表明，SMC5/6 以复制依赖性方式被招募到质粒底物上。重要的是，我们用新开发的、无偏见的蛋白质组学方法检测质粒结合蛋白。这使我们能够准确量化所有 SMC5/6 亚基以及粘连蛋白和所有 DNA 复制因子。使用这个独特的系统，我们将解决以下问题：1) SMC5/6 何时以及如何加载到 DNA 上？我们将在正常和扰动的 DNA 复制过程中监测 DNA 中的蛋白质募集。我们将通过识别相关的 DNA 底物和负载复合物来确定调节 SMC5/6 向 DNA 的生理募集的细胞网络。我们将测试 SMC5/6 蛋白是否形成介导 DNA 拓扑结合的环状结构。我们进一步提出实验来深入了解 SMC5/6 的独特结构及其 ATP 酶、相扑和泛素 E3 连接酶活性与其功能的关系。 2）SMC5/6的功能是什么？我们将生产用于从非洲爪蟾卵提取物中消耗SMC5/6或其负载因子的试剂以及用于救援实验的重组蛋白。使用具有确定质粒底物的耗尽提取物，我们将使用各种已建立的测定法来分析 DNA 复制和修复中间体。这些实验将直接揭示 SMC5/6 在复制产物的串联或确定的 DNA 修复途径的不同步骤中的可能功能。通过将生化上易于处理的系统与确定的 DNA 底物和公正、超灵敏的蛋白质组学方法相结合，我们将首次能够将 DNA 中间体的时间出现与 DNA 中涉及的分子机器的组装关联起来。 交易。我们预计这些方法将为迄今为止神秘的 SMC5/6 复合物的功能和作用提供前所未有的见解。

项目成果

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection

Nse5/6 抑制 Smc5/6 ATPase 以促进 DNA 底物选择

• DOI: 10.1101/2021.02.09.430422
• 发表时间: 2021
• 期刊: bioRxiv
• 作者: Michael Taschner;Jérôme Basquin;Barbara Steigenberger;Ingmar Schaefer;Young-Min Soh;Claire Basquin;Esben Lorentzen;Markus Räschle;Richard A. Scheltema;Stephan Gruber
• 通讯作者: Stephan Gruber

Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts

• DOI: 10.1016/j.molcel.2018.11.024
• 发表时间: 2019-02-07
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Larsen, Nicolai B.;Gao, Alan O.;Duxin, Julien P.
• 通讯作者: Duxin, Julien P.

The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair

• DOI: 10.1016/j.cell.2018.10.053
• 发表时间: 2019-01-10
• 期刊: CELL
• 影响因子: 64.5
• 作者: Sparks, Justin L.;Chistol, Gheorghe;Walter, Johannes C.
• 通讯作者: Walter, Johannes C.