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.

我们的身体由数万亿个细胞组成。随着时间的推移，我们的细胞会老化并受损，因此我们体内的一部分细胞会不断生长和分裂，创造出自己的替代品。在每次细胞分裂之前，每个细胞都必须首先复制它的DNA -所有的DNA，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，进而导致细胞增殖、衰老和癌症发展的问题。大多数癌症驱动突变是DNA复制过程中随机错误的结果。此外，DNA复制机制组成部分的遗传突变导致一系列以身材矮小和大脑小为特征的疾病，这是由于无法产生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，高效而精确地复制，然后再次压缩。为了复制所有的DNA，DNA复制的过程从大约5万个起始位点（复制起点）开始，在这个过程中，一些起点在早期被激活，一些在晚期被激活。起点激活的过程已经被很好地表征，并从一个简单的真核生物-面包酵母中的纯化蛋白质中重建，因此定义了完成起点激活所需的最小蛋白质组。然而，在包括人类在内的更复杂的生物体中，有几个参与者仍然未知。我们在准备这一提议时产生的数据表明，在酵母中不存在的蛋白质DONSON可能是酵母关键来源激活剂之一Sld 2的功能等价物，而在高等真核生物中缺少这种等价物。当DONSON突变时，会导致Meier-Gorlin综合征-一种由错误的DNA复制启动引起的侏儒症;相反，它的过度表达与几种癌症类型的发展有关。DONSON已被证明对维持DNA复制很重要，但其分子功能尚未确定，也没有描述DONSON在起始激活中的作用。在这里，我们建议调查DONSON在两个高等真核生物模型系统中的DNA复制起始过程中的功能：从非洲爪蛙的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物化学方法来了解DONSON在时间上在起源激活过程中的位置：它与哪些其他激活剂相互作用，它在过程中的哪个特定步骤中发挥作用，以及没有DONSON的起源及其激活剂会发生什么。我们还将确定DONSON的哪个部分对其功能很重要，以及它如何被驱动起源激活的酶-细胞周期蛋白依赖性激酶（CDK）调节。所有这些都将确定DONSON是否可以作为复制起点的关键激活剂。在一个独立的研究路径中，我们将确定DONSON是否在人类永生化细胞系的起点激活中发挥作用。我们已经使用基因组编辑技术来修饰细胞内的DONSON，使其与降解标签融合，该降解标签通过向细胞培养物中添加植物激素（生长素）来激活（生长素诱导的降解决定子，AID）。这种方法允许在添加生长素时快速降解（通常在30-60分钟内）目的蛋白。我们最近使用AID系统结合细胞同步技术来发现另一种参与复制的蛋白质TRAIP的功能。我们现在将遵循与DONSON类似的研究路径：我们将使用生物化学，显微镜，单分子和全基因组方法确定DONSON降解对起源发射的后果。