The role of DONSON during DNA replication initiation

我们的身体是由数万亿个细胞组成的。随着时间的推移，我们的细胞老化并受损，所以我们体内的一部分细胞继续生长和分裂，产生自己的替代品。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不能及时修复，就会导致突变和基因变化，进而导致细胞增殖、衰老和癌症的发生。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，由于无法产生足够的细胞来发育正常大小的人，DNA复制机制组成部分的遗传突变会导致一系列以身材矮小和大脑小为特征的疾病。复制我们所有的DNA是一项艰巨的任务——我们每个细胞中大约有2米长的DNA，它以一种高度有序的方式被压缩，以适应细胞核，从而使蛋白质能够访问任何所需的DNA序列。在DNA复制过程中，这种结构必须展开，高效而精确地复制，然后再次压缩。为了复制所有的DNA， DNA的复制过程从大约5万个起始位点（复制起点）开始，在这个过程中，有些起始位点被激活得早，有些被激活得晚。起源激活的过程已经被很好地表征，并从一个简单的真核生物，面包师酵母中纯化的蛋白质中重构，因此定义了实现起源激活所需的最小蛋白质集。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。我们在准备本提案时生成的数据表明，酵母中不存在的蛋白质DONSON可能是酵母关键起源激活因子之一Sld2的功能等价物，而在高等真核生物中缺少等价物。当DONSON基因发生突变时，会导致Meier-Gorlin综合征——一种由错误的DNA复制起始引起的侏儒症；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在起源激活中的作用。在这里，我们建议研究DONSON在两种高级真核模型系统中DNA复制起始的功能：从非洲爪蛙卵中制备的无细胞提取物和永生的人类细胞系。我们将在蛋提取物中使用生化方法来了解DONSON在起源激活过程中的暂时位置：它与哪些其他活化剂相互作用，它在该过程中的哪个特定步骤中发挥作用，以及没有DONSON的起源及其活化剂会发生什么。我们还将确定DONSON的哪个部分对其功能很重要，以及它如何受到驱动起源激活的酶-周期蛋白依赖激酶（CDKs）的调节。所有这些都将证明DONSON是否可以作为复制起始的关键激活因子。在独立的研究路径中，我们将确定DONSON是否在人类永生细胞系的起源激活中起作用。我们使用基因组编辑技术修改细胞内的DONSON，使其与降解标签融合，该降解标签通过在细胞培养物中添加植物激素（生长素）来激活（生长素诱导Degron， AID）。这种方法允许在添加生长素后快速降解（通常在30-60分钟内）感兴趣的蛋白质。我们最近使用AID系统结合细胞同步技术发现了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：我们将使用生化，显微镜，单分子和全基因组方法确定DONSON降解对原点着火的影响。

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.