The role of DONSON during DNA replication initiation

DONSON 在 DNA 复制起始过程中的作用

• 批准号: BB/Y002458/1
• 负责人: Agnieszka Gambus
• 金额: $ 79.51万
• 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%2FY002458%2F1
• 关键词: role; DONSON; during; DNA; replication

Our bodies are built of trillions of cells. Over time, our cells age and become damaged, so a subset of cells in our bodies keep growing and dividing, creating their own replacements. Before each cell division, every cell must first duplicate its DNA - all of it, just once and without mistakes. Mistakes during DNA replication that 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 are results of 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 stature and small brain due to inability to create enough cells to develop a normally sized human being. To replicate all 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 with efficiency and precision, and compacted again. To replicate all DNA, the process of DNA replication starts from about 50 thousand start sites (origins of replication), with some origins being activated early and some late during the process.The process of origin activation has been well characterised and reconstituted from purified proteins in a simple eukaryotic organism, bakers' yeast, therefore defining the minimal set of proteins needed to fulfil origin activation. However, in more complex organisms, including humans, several players remain unknown. The data we generated in preparation of this proposal suggest that a protein DONSON, which does not exist in yeast, may be a functional equivalent of one of the yeast key origin activators, Sld2, for which such an equivalent in higher eukaryotes is missing. DONSON, when mutated, leads to Meier-Gorlin syndrome - a dwarfism disorder caused by faulty DNA replication initiation; conversely, its overexpression is linked with development of several cancer types. DONSON has been shown to be important for sustaining DNA replication, but its molecular function has not been determined and there is no described role for DONSON in origin activation. Here we propose to investigate the function of DONSON during DNA replication initiation in two higher eukaryotic model systems: cell-free extract prepared from African Clawed frog's eggs and immortalised human cell lines. We will use biochemical approaches in egg extract to understand where DONSON temporally fits within the origin activation process: which other activators it interacts with, which specific step in the process it plays a role in, and what happens to origins and their activators without DONSON. We will also determine which part of DONSON is important for its function and how it is regulated by enzymes driving origin activation - cyclin dependent kinases (CDKs). All these will establish if DONSON can act as a key activator of replication origins.In an independent path of investigation, we will determine if DONSON plays a role in origin activation in human immortalised cell lines. We have used genome editing techniques to modify DONSON within cells to fuse it with a degradation tag, which is activated by addition of a plant hormone (auxin) to the cell culture (Auxin Induced Degron, AID). This approach allows for rapid degradation (usually within 30-60 min) of the protein of interest upon auxin addition. We have recently used the AID system combined with cell synchronisation techniques to discover the function of another protein involved in replication - TRAIP. We will now follow an analogous path of investigation with DONSON: we will determine the consequences of DONSON degradation for origin firing using biochemical, microscopy, single-molecule and genome-wide approaches.

我们的身体是由数万亿个细胞建造的。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成长和分裂，创造了自己的替代品。在每个细胞分裂之前，每个细胞都必须首先复制其DNA - 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在DNA复制过程中随机错误的结果。此外，DNA复制机制的组成部分中的遗传突变导致一组具有小大脑和小脑的疾病，因为无法创建足够的细胞来发展正常大小的人。复制我们所有的DNA是一项艰巨的任务 - 我们每个细胞中有大约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需要的DNA序列的方式适合细胞核。在DNA复制过程中，必须解开此结构，以效率和精度重复，然后再次压实。要复制所有DNA，DNA复制的过程从大约50,000个起始位点（复制的起源）开始，其中一些起源在过程中被激活，有些是在过程中激活的后期激活的过程，在一个简单的真核生物有机体中，从纯化的蛋白质中进行了很好的表征和重新构成，因此，面包师的Yeast，Bakery YEAST，酵母的Yeast需要实现的原始蛋白质，以实现蛋白质的激活。但是，在包括人类在内的更复杂的生物体中，几个玩家仍然未知。我们在准备该提案的数据中生成的数据表明，酵母中不存在的蛋白质唐森可能是酵母钥匙来源激活剂SLD2的功能相当的功能，因为该激活剂SLD2缺失了这种等同于较高的真核生物。唐森（Donson）发生突变时会导致Meier -Gorlin综合征 - 由DNA复制启动故障引起的矮症。相反，其过表达与几种癌症类型的发展有关。唐森已被证明对于维持DNA复制很重要，但是尚未确定其分子功能，并且唐森在起源激活中没有描述的作用。在这里，我们建议在两个较高的真核模型系统中研究唐森在DNA复制启动过程中的功能：由非洲爪的青蛙卵和永生化的人类细胞系制备的无细胞提取物。我们将在鸡蛋提取物中使用生化方法来了解唐森在临时激活过程中适合何处：它与哪些其他激活剂相互作用，在该过程中发挥作用的哪个特定步骤，以及没有唐森的原始及其激活剂发生的事情。我们还将确定唐森的哪一部分对其功能很重要，以及如何通过驱动起源激活的酶调节 - 细胞周期蛋白依赖性激酶（CDKS）。所有这些都将确定唐森是否可以充当复制起源的关键激活因子。在独立的研究途径中，我们将确定唐森在人生永生细胞系中是否在起源激活中起作用。我们已经使用基因组编辑技术将细胞内的唐森修饰与降解标签融合，该标签通过在细胞培养物中添加植物激素（Auxin）（生长素诱导的DEGRON，AID）激活。这种方法允许加入生长素后快速降解（通常在30-60分钟内）。我们最近使用了与细胞同步技术相结合的辅助系统来发现与复制 - TRAIP有关的另一种蛋白质的功能。现在，我们将遵循与唐森的类似研究路径：我们将使用生化，显微镜，单分子和全基因组方法来确定唐森退化对起源发射的后果。