DNA Recruitment, Regulation & Function of SMC5/6

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（粘附素、凝聚素和SMC 5/6）发挥重要作用。凝聚素和凝聚素形成包围DNA双链体的大环。虽然凝聚素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-凝聚素促进染色体压缩和遗传物质的一个拷贝精确分离到子细胞。然而，SMC 5/6的确切功能仍然未知。SMC 5/6的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。因此，SMC 5/6功能受损会加速动物模型中的肿瘤发生。我们的初步研究结果表明，SMC 5/6被招募到质粒基板在复制依赖的方式。重要的是，我们检测质粒结合蛋白质与新开发的，公正的蛋白质组学方法。这使得我们能够准确地定量所有的SMC 5/6亚基以及粘附素和所有的DNA复制因子。使用这个独特的系统，我们将解决以下问题：1）何时以及如何将SMC 5/6加载到DNA上？我们将监测蛋白质招募DNA在正常和扰动DNA复制。我们将通过鉴定相关的DNA底物和负载复合物来确定调节SMC 5/6向DNA的生理募集的细胞网络。我们将测试SMC 5/6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SMC 5/6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)SMC 5/6的功能是什么？我们将从非洲爪蟾卵提取物中产生用于耗尽SMC 5/6或其加载因子的试剂，以及用于拯救实验的重组蛋白。使用含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分析DNA复制和修复中间体。这些实验将直接揭示SMC 5/6在复制产物脱链或DNA修复途径的不同步骤中的可能功能，通过将生物化学上易于处理的系统与定义的DNA底物和无偏见的超灵敏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相关联。我们预计，这些方法将提供前所未有的深入了解迄今为止神秘的SMC 5/6复合物的功能和作用。

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.