Cryo-EM studies of a metazoan replisome captured ex vivo during elongation and termination

我们的身体由数万亿个细胞组成。随着时间的推移，我们的细胞老化并受损，因此我们体内的一部分细胞不断分裂，产生替代品。在每次细胞分裂之前，每个细胞都必须首先复制它的基因组-所有的基因组，只有一次，没有错误。DNA复制过程中的错误，如果没有及时修复，可能会导致突变和遗传变化，从而导致细胞增殖，衰老和癌症发展的问题。大多数癌症驱动突变是由DNA复制过程中的随机错误引起的。此外，DNA复制机制组件的遗传突变导致一系列以小姿势和小大脑为特征的疾病，这是由于无法创造足够的细胞来发育正常大小的人类。复制我们所有的DNA是一项艰巨的任务-我们每个细胞中大约有2米长的DNA，它以高度组织的方式压缩，以适应细胞核，使蛋白质能够访问任何需要的DNA序列。在DNA复制过程中，这种结构必须被解开、复制和再次压缩。为了复制所有的DNA，DNA复制过程从大约5万个起始位点开始，大约有10万个复制机器（复制体）复制DNA。自70年前沃森和克里克提出第一个DNA复制模型以来，研究人员一直致力于了解这一过程是如何协调、调节和无错误地传递的。在真核细胞中，复制机制由数百种蛋白质组成，这些蛋白质必须精确地组织起来才能协调它们的所有功能。我们以前的工作表明，复制体的核心是围绕复制解旋酶（CMG复合物）组织的。复制解旋酶可以解旋双链DNA，为互补链的合成提供模板。在过去的15年里，结构生物学的发现已经产生了重组解旋酶的第一个结构，为我们理解复制机制的一些组件如何协同工作提供了重大突破。然而，几乎所有的解决复合物组装在体外从纯化的蛋白质。这种方法显然是非常成功的，但它需要预先确定的已知因素，这些因素被假定为形成感兴趣的复合体，可能会遗漏可能影响复合体整体结构的额外或次要合作伙伴。此外，参与这些过程的分子机器自然组装在染色质化的底物上，并受到严格的调控。由于重组复合物是在体外组装的，因此缺少该调节的元素，从而可能导致不完整或误导性的观察结果。最后，大多数解析的结构都是从芽殖酵母蛋白质中重建的，这些蛋白质与人类或其他高等真核生物的蛋白质不同。在这里，我们建议优化一种替代方法来分离蛋白质复合物的DNA复制所必需的非洲爪蟾卵提取物，这是唯一的高等真核生物无细胞系统包含所有参与DNA复制的因素。纯化的蛋白质复合物将通过结构显微镜技术和生物化学方法进行分析，提供第一个自然（离体）组装的复制解旋酶和复制体的结构。我们将对我们的结构进行生物化学验证，并将其与其他物种现有的体外组装结构进行比较。此外，利用我们对这个系统的专业知识，我们可以使用各种抑制剂来“冻结”各种配置的复制机制：活跃的，停滞的，终止的。我们将解析它们的结构并进行比较，以了解复制体在这些状态转换时发生的动态变化。

Our bodies are built-up of trillions of cells. Over time, our cells age and become damaged, so a subset of cells in our bodies keep dividing, creating replacements. Before each cell division, every cell must first duplicate its genome - all of it, just once and without mistakes. Mistakes during DNA replication, which are not timely repaired, can lead to mutations and genetic changes that in turn can lead to problems with cell proliferation, aging, and development of cancer. Most of the cancer-driving mutations result from random mistakes during the process of DNA replication. Moreover, hereditary mutations in components of the DNA replication machinery cause a set of disorders characterised by small posture and small brain due to the inability to create enough cells to develop a normal-sized human being. Replicating all of our DNA is a huge task - we have about 2 metres of DNA in each of our cells, and it is compacted in a highly organised way to fit into the nucleus in a manner that enables proteins to access any needed DNA sequences. During DNA replication this structure must be unwound, duplicated, and compacted again. To replicate all DNA, the process of DNA replication starts from about 50 thousand start sites with about 100 thousand individual replication machineries (replisomes) replicating DNA. Ever since Watson and Crick proposed the first model of DNA replication 70 years ago, researchers aim to understand how this process is coordinated, regulated, and delivered without mistakes.In eukaryotic cells, the replication machinery is composed of hundreds of proteins that must be precisely organised to coordinate all their functions together. Our previous work has shown that the core of the replisome is organised around the replicative helicase (CMG complex). The replicative helicase can unwind double-stranded DNA to provide the template for synthesis of the complementary strands. Over the last 15 years, structural biology findings have produced the first structures of reconstituted helicase providing a great breakthrough into our understanding of how some of the components of the replication machinery are working together. However, almost all the solved complexes were assembled in vitro from purified proteins. This approach is obviously very successful, but it requires pre-determined known factors that are assumed to form the complex of interest, potentially missing additional or minor partners that could affect the overall structure of the complex. Moreover, the molecular machineries involved in these processes are naturally assembled on a chromatinised substrate and are tightly regulated. Since reconstituted complexes are assembled in vitro, elements of that regulation are missing, thus potentially leading to incomplete or misleading observations. Finally, most of the solved structures are reconstituted from budding yeast proteins, which are not identical to proteins from human or other higher eukaryotic organisms. We propose here to optimize an alternative method to isolate protein complexes essential for DNA replication using Xenopus laevis egg extract, which is the only higher eukaryote cell-free system containing all the factors involved in DNA replication. The purified protein complexes will be analysed via structural microscopy techniques and biochemical approaches delivering the first ever naturally (ex vivo) assembled structures of a replicative helicase and the replisome. We will biochemically validate our structures and compare them to the existing in vitro assembled structures from other species. Moreover, using our expertise of working with this system, we can use various inhibitors to "freeze" the replication machinery in various configurations: active, stalled, terminated. We will solve their structures and compare them, to understand the dynamic changes that occur to the replisome as it transitions through these states.