Roles of ubiquitin and SUMO during chromosomal DNA replication.

细胞分裂是生命繁殖的基础。这涉及到基因信息的精确复制，这被称为DNA复制。这一过程必须得到，而且已经得到了精确的规范。任何没有随后修复的错误都可能改变细胞的行为方式，导致遗传病、癌症和衰老等疾病。令人着迷的是，在大多数情况下，细胞可以完成这项任务，精确地复制整个基因组一次，没有错误。基因组复制对于癌细胞的快速分裂也是必不可少的。这就是为什么许多抗癌疗法都以DNA复制为目标，但到目前为止，这些疗法并不是针对癌细胞的，而且有明显的副作用。因此，至关重要的是，我们必须充分了解这一基本过程，这样我们才能制定出更好、更具体的抗癌策略。细胞已经发展出许多方法来处理受损的DNA，以保持完整的基因组。最近几年，人们发现了小蛋白修饰剂泛素和SUMO在调节关键DNA修复蛋白中的重要性。有强有力的证据表明，这些修饰在未受损DNA的复制过程中也很重要。然而，人们对它们所针对的蛋白质知之甚少。我项目的目的是研究泛素和SUMO在未损伤DNA复制过程中的作用。两个生物系统将被用来完成这个项目。使用非洲爪蛙（Xenopus laevis）卵提取物的无细胞系统含有细胞周期进程所需的大多数预先形成的蛋白质复合物，因此可以在试管中支持完整的一轮DNA复制。由于这一过程的许多方面在所有研究的真核生物中都是高度相似的，因此在更简单的蛋提取物模型系统中确定的机制通常也适用于人类细胞。一旦发现了新的修饰，并对爪蟾卵提取物进行了基本的机制研究，我想研究类似的机制是否也适用于人类细胞。根据我的初步数据，我特别感兴趣的是研究泛素在DNA复制终止阶段的作用。当来自染色体相反位置的两个DNA复制叉融合在一起时，复制终止就发生了。这些结构的有效和完美的解析对于维持基因组的完整性至关重要。人类细胞中有成千上万的复制分叉，在每个终止阶段都必须解决——因此，我们创造工具来研究这一过程并确定其对肿瘤发生的影响是至关重要的。然而，这一阶段的DNA复制也很少被理解，因此是一个非常令人兴奋的研究领域。我将研究泛素化调节这一过程的机制及其破坏的后果。以类似的方式，我将研究阻断sumo化对DNA复制的不同方面的影响。我还想利用非洲爪蟾系统DNA复制生物化学分析的简单性，对与复制DNA相关的蛋白质和DNA复制过程中的修饰蛋白进行系统的分析。然后，我将选择最有趣的，并描述这些修改的类型和地点。我的最终目的是确定鉴定修饰的功能及其在DNA复制和癌症发展过程中的重要性。定义泛素和SUMO修饰在DNA复制中的作用将扩大我们对这一过程的理解。DNA复制和泛素系统都是目前许多抗癌化疗的目标。因此，揭示这两者之间的新串扰途径可能提示新的靶点或化疗药物的组合，因为它可能揭示产生对癌细胞特异性致命的DNA损伤的新方法。

Cell division is the basis for the propagation of life. This involves the precise duplication of genetic information, which is called DNA replication. This process must be, and is, precisely regulated. Any mistakes that are not subsequently repaired can change the way the cell behaves and result in conditions such as genetic diseases, cancer and ageing. It is fascinating that in most cases cells can achieve this task of duplicating the whole genome precisely once and without mistakes. Genome duplication is also essential for fast dividing cancer cells. It is why many anti-cancer therapies target DNA replication, but these are so far not specific for cancer cells and have significant side-effects. It is therefore crucial that we fully understand this fundamental process so that we can develop better and more specific anti-cancer strategies. Cells have developed many ways of dealing with damaged DNA to maintain an intact genome. Last few years brought to light the importance of small-protein modifiers called ubiquitin and SUMO in regulating key DNA repair proteins. There is strong evidence to suggest that these modifications are also important during the replication of undamaged DNA. However little is known about the proteins they are targeting. The aim of my project is to investigate the role of ubiquitin and SUMO during replication of undamaged DNA.Two biological systems will be used to fulfill this project. The cell-free system using extracts from the eggs of the African clawed frog (Xenopus laevis) contains pre-formed complexes of most proteins required for cell cycle progression and so can support a complete round of DNA replication in a test tube. As many aspects of this process are highly similar in all eukaryotic organisms studied, the mechanisms identified in simpler egg extract model system are most often true also in human cells. Once novel modifications have been identified and basic mechanistic studies have been performed in Xenopus egg extract I would like to investigate whether analogous mechanism function also in human cells.In particular, I am interested in investigating the role of ubiquitin during the termination stage of DNA replication, as suggested by my preliminary data. Replication termination occurs when two DNA replication forks coming from the opposite sites of the chromosome fuse together. The efficient and faultless resolution of these structures is crucial for maintaining the genome integrity. There are thousands of replication forks in human cell which have to be resolved during each termination phase - it is crucial therefore that we create tools to study this process and determine its input towards tumorgenesis. However, this stage of DNA replication is also very poorly understood and therefore a very exciting research area. I will study the mechanism by which ubiquitylation regulates this process and the consequences of its disruption. In an analogous way, I will examine the effects of blocking sumoylation on different aspects of DNA replication.I would also like to take advantage of the simplicity of biochemical analysis of DNA replication in Xenopus system and perform a systematic analysis of proteins associating with replicating DNA and modified during DNA replication. I will then choose the most interesting ones and characterize the type and site of these modifications. My final aim is to determine the function of the identified modifications and their importance in process of DNA replication and cancer development.Defining the role of ubiquitin and SUMO modifications during DNA replication will widen our understanding of this process. Both DNA replication and the ubiquitin system are targeted by many current anti-cancer chemotherapies. Unveiling new crosstalk pathways between these two may therefore suggest novel targets or combinations of chemotherapeutic agents as it may reveal new ways of creating DNA damage specifically lethal to cancer cells.