DNA Recruitment, Regulation & Function of SMC5/6

复杂的调控机制控制着染色质结构。因此，SMC家族的蛋白复合物（黏结蛋白、凝聚蛋白和SMC5/6）起着至关重要的作用。内聚蛋白和凝聚蛋白形成包含DNA双链的大环。当内聚蛋白将姐妹染色单体保持在一起时——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的紧实和将遗传物质的一个拷贝精确地分离到子细胞。相比之下，SMC5/6的确切功能仍然未知。SMC5/6的失活分别导致有丝分裂中的细胞周期立即停止或遗传物质向子细胞的不准确传递。一致地，在动物模型中，受损的SMC5/6功能加速了肿瘤的发生。本研究拟研究SMC5/6在爪蟾卵提取物中的功能。我们的初步结果表明SMC5/6以复制依赖的方式被招募到质粒底物上。重要的是，我们用新开发的无偏蛋白质组学方法检测质粒结合蛋白。这使我们能够准确地定量所有SMC5/6亚基以及黏结蛋白和所有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？我们将在正常和受干扰的DNA复制过程中监测蛋白质向DNA的招募。我们将通过鉴定相关的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蛋白是否形成环状结构，介导DNA的拓扑结合。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，sumo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？我们将从爪蟾卵提取液中提取SMC5/6或其装载因子，并制备用于救援实验的重组蛋白。使用枯竭提取物与定义质粒底物，我们将分析DNA复制和修复中间体使用各种既定的测定。这些实验将直接揭示SMC5/6在复制产物的十烷化或定义的DNA修复途径的不同步骤中的可能功能。通过将生物化学可处理的系统与定义的DNA底物和无偏、超灵敏的蛋白质组学方法相结合，我们将首次能够将DNA中间体的时间外观与参与DNA交易的分子机器的组装联系起来。我们预计这些方法将为迄今为止神秘的SMC5/6复合体的功能和作用提供前所未有的见解。

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.