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

在延伸和终止过程中离体捕获的后生动物复制体的冷冻电镜研究

• 批准号: BB/Y006232/1
• 负责人: Agnieszka Gambus
• 金额: $ 67.93万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY006232%2F1
• 关键词: Cryo; EM; studies; metazoan; replisome

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.

我们的身体建立了数万亿个细胞。随着时间的流逝，我们的细胞会变老并受损，因此我们体内的一部分细胞不断分裂，从而造成更换。在每个细胞分裂之前，每个细胞都必须首先复制其基因组 - 所有细胞，仅一次，没有错误。在DNA复制过程中的错误（未及时修复）会导致突变和遗传变化，这反过来又可能导致细胞增殖，衰老和癌症的发育问题。大多数癌症驱动突变是由DNA复制过程中随机错误引起的。此外，由于无法创建足够的细胞来发展正常大小的人，因此DNA复制机制成分的遗传突变引起了一组具有小姿势和小脑的疾病。复制我们所有的DNA是一项艰巨的任务 - 我们每个细胞中都有大约2米的DNA，并且以高度组织的方式压实，以使蛋白质能够访问任何必要的DNA序列的方式适合细胞核。在DNA复制过程中，必须重新解开，重复并再次压实该结构。为了复制所有DNA，DNA复制的过程从约50,000个启动位点开始，大约有1万个单独的复制机器（重新组合）复制DNA。自从沃森（Watson）和克里克（Crick）提出了70年前DNA复制的第一个模型以来，研究人员的目标是了解该过程是如何协调，调节和没有错误的。在真核细胞中，复制机制由数百种必须精确组织的蛋白质组成，以使其所有功能协调在一起。我们以前的工作表明，重置体的核心是围绕复制旋转酶（CMG复合物）组织的。复制性解旋酶可以放松双链DNA，以提供互补链合成的模板。在过去的15年中，结构生物学发现产生了重构解旋酶的第一个结构，这为我们对复制机制的某些组成部分的理解提供了重大突破。但是，几乎所有溶解的复合物都是从纯化蛋白质中组装的。这种方法显然非常成功，但是它需要假定构成感兴趣的复杂的预定的已知因素，这些因素可能缺少可能影响复合物的整体结构的其他或次要伴侣。此外，这些过程中涉及的分子机器自然组装在染色质底物上，并受到严格调节。由于重构的复合物是在体外组装的，因此缺少该调节的元素，因此可能导致不完整或误导性观察结果。最后，大多数解决的结构都是从萌芽的酵母蛋白中重构的，酵母蛋白与人类或其他高等真核生物的蛋白质并不相同。我们在这里建议使用Xenopus laevis Egg提取物进行优化一种用于DNA复制所必需的蛋白质复合物的替代方法，这是唯一包含所有涉及DNA复制因素的较高的无真核细胞系统。纯化的蛋白质复合物将通过结构显微镜技术和生化方法进行分析，从而提供了有史以来的第一种自然（Ex vivo）组装结构的复制性解旋酶和重壳体的结构。我们将在生物化学上验证我们的结构，并将其与其他物种的现有体外组装结构进行比较。此外，利用我们与该系统合作的专业知识，我们可以使用各种抑制剂来“冻结”各种配置中的复制机制：主动，停滞，停滞，终止。我们将解决它们的结构并进行比较，以了解重新构体通过这些状态过渡时发生的动态变化。