Roles of ubiquitin and SUMO during chromosomal DNA replication.

细胞分裂是生命繁殖的基础。这涉及到遗传信息的精确复制，这称为DNA复制。这一过程必须而且正在受到严格的监管。任何后来没有修复的错误都可能改变细胞的行为方式，并导致遗传病、癌症和衰老等疾病。令人着迷的是，在大多数情况下，细胞可以一次准确地完成复制整个基因组的任务，而不会出错。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针对DNA复制，但到目前为止，这些疗法并不是针对癌细胞的，而且有显著的副作用。因此，我们必须充分了解这一基本过程，以便我们能够制定更好和更具体的抗癌战略。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。过去几年揭示了被称为泛素和相扑的小蛋白修饰物在调节关键DNA修复蛋白方面的重要性。有强有力的证据表明，这些修饰在复制未受损的DNA过程中也很重要。然而，人们对他们所针对的蛋白质知之甚少。我的项目的目的是研究泛素和相扑在复制未受损DNA过程中的作用。将使用两个生物系统来完成这一项目。使用非洲爪蛙(非洲爪蛙)卵提取物的无细胞系统包含细胞周期进展所需的大多数蛋白质的预先形成的复合体，因此可以支持试管中的完整一轮DNA复制。由于这一过程的许多方面在所有被研究的真核生物中都高度相似，在更简单的鸡蛋提取模型系统中发现的机制通常也适用于人类细胞。一旦确定了新的修饰并对非洲爪哇卵提取液进行了基础机制研究，我想调查类似的机制是否也在人类细胞中发挥作用，特别是我的初步数据表明，我对研究泛素在DNA复制终止阶段的作用感兴趣。当来自染色体相反位置的两个DNA复制叉融合在一起时，复制终止发生。这些结构的有效和完美的分辨对于保持基因组的完整性是至关重要的。人类细胞中有数以千计的复制分叉，在每个终止阶段都必须解决-因此，我们创建工具来研究这一过程并确定其对肿瘤发生的输入是至关重要的。然而，对DNA复制的这一阶段也知之甚少，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中断的后果。以类似的方式，我将研究阻断相扑作用对DNA复制的不同方面的影响。我还想利用非洲爪哇系统中DNA复制的生物化学分析的简便性，对与DNA复制相关的蛋白质和DNA复制过程中的修饰进行系统的分析。然后，我将选择最有趣的修改，并描述这些修改的类型和位置。我的最终目标是确定已识别的修饰的功能及其在DNA复制和癌症发展过程中的重要性。确定泛素和相扑修饰在DNA复制过程中的作用将拓宽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靶点。因此，揭示这两者之间的新串扰通路可能会提出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别致命的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.