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，我们的细胞会老化并受到破坏，因此我们体内的一部分细胞会继续生长和分裂，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自己的DNA--所有的DNA，只复制一次，并且没有错误。DNA复制过程中的错误如果得不到及时修复，可能会导致突变和基因变化，进而可能导致细胞增殖、衰老和癌症发展的问题。大多数致癌突变是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会导致一系列疾病，其特征是身材矮小，大脑小，因为无法创造足够的细胞来发育正常大小的人。复制我们所有的DNA是一项艰巨的任务--我们每个细胞中都有大约2米长的DNA，它以一种高度有组织的方式被压缩，以适应细胞核的方式，使蛋白质能够访问任何所需的DNA序列。在DNA复制过程中，这个结构必须解开，以高效和精确的方式复制，然后再次压缩。为了复制所有的DNA，DNA的复制过程从大约5万个起点(复制起点)开始，一些起点在复制过程中被提前激活，另一些在过程中后期被激活。起源激活过程已经被很好地描述，并由简单的真核生物面包师酵母中的纯化蛋白重组，因此定义了实现起点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。我们在准备这项提案时产生的数据表明，酵母中不存在的一种蛋白质Donson可能是酵母关键起源激活子之一SLD2的功能等价物，而在高等真核生物中缺少这样的等价物。当Donson基因突变时，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的发生有关。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，我们打算在两个更高级的真核模型系统中研究Donson在DNA复制启动中的作用：从非洲爪蛙卵制备的无细胞提取物和永生化的人类细胞系。我们将在鸡蛋提取物中使用生化方法来了解Donson在起源激活过程中的临时位置：它与其他哪些激活剂相互作用，它在这个过程中发挥作用的具体步骤，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是重要的，以及它是如何由驱动起始激活的酶-细胞周期蛋白依赖激酶(CDK)调节的。所有这些都将确定Donson是否可以作为复制起源的关键激活剂。在一条独立的调查路径中，我们将确定Donson是否在人类永生化细胞系的起源激活中发挥作用。我们使用基因组编辑技术在细胞内修饰Donson，将其与一个降解标签融合，该标签通过在细胞培养中添加植物激素(生长素)来激活(生长素诱导的Degron，AID)。这种方法允许生长素添加后目标蛋白质的快速降解(通常在30-60分钟内)。我们最近使用了AID系统和细胞同步技术来发现另一种参与复制的蛋白质-TRAIP的功能。我们现在将对唐森进行类似的研究：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降解对起火的后果。

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.