Roles of ubiquitin and SUMO during chromosomal DNA replication.

泛素和 SUMO 在染色体 DNA 复制过程中的作用。

• 批准号: MR/K007106/1
• 负责人: Agnieszka Gambus
• 金额: $ 141.38万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FK007106%2F1
• 关键词: Roles; ubiquitin; SUMO; during; chromosomal

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.

细胞分裂是生命繁衍的基础。这涉及遗传信息的精确复制，称为DNA复制。这个过程必须并且确实受到精确监管。任何随后未修复的错误都可能改变细胞的行为方式并导致遗传疾病、癌症和衰老等疾病。令人着迷的是，在大多数情况下，细胞可以完成一次精确复制整个基因组的任务，而且不会出现错误。基因组复制对于快速分裂的癌细胞也至关重要。这就是为什么许多抗癌疗法都以 DNA 复制为目标，但迄今为止这些疗法都不是针对癌细胞的，并且具有显着的副作用。因此，我们充分了解这一基本过程至关重要，这样我们才能制定更好、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，以维持完整的基因组。过去几年，人们认识到泛素和 SUMO 小蛋白修饰剂在调节关键 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。我的项目的目的是研究泛素和 SUMO 在未受损 DNA 复制过程中的作用。将使用两个生物系统来完成该项目。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展所需的大多数蛋白质的预形成复合物，因此可以支持试管中完整一轮的 DNA 复制。由于该过程的许多方面在所有研究的真核生物中都高度相似，因此在更简单的鸡蛋提取物模型系统中确定的机制在人类细胞中也通常是正确的。一旦确定了新的修饰并在非洲爪蟾卵提取物中进行了基本机制研究，我想研究类似的机制是否也在人类细胞中发挥作用。特别是，我对研究泛素在 DNA 复制终止阶段的作用感兴趣，正如我的初步数据所示。当来自染色体相对位点的两个 DNA 复制叉融合在一起时，就会发生复制终止。这些结构的高效且完美的解析对于维持基因组完整性至关重要。人类细胞中有数千个复制叉，必须在每个终止阶段得到解决 - 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至关重要。然而，人们对 DNA 复制的这一阶段知之甚少，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破坏的后果。以类似的方式，我将研究阻断苏酰化对 DNA 复制不同方面的影响。我还想利用非洲爪蟾系统中 DNA 复制生化分析的简单性，对与 DNA 复制相关的蛋白质进行系统分析并在 DNA 复制过程中进行修饰。然后我将选择最有趣的，并描述这些修改的类型和地点。我的最终目标是确定已识别修饰的功能及其在 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当前许多抗癌化疗的目标。因此，揭示这两者之间新的串扰途径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。

项目成果

Mechanisms of eukaryotic replisome disassembly.

真核复制体拆卸的机制。

• DOI: http://dx.10.1042/bst20190363
• 发表时间: 2020
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Moreno SP

Two paths to let the replisome go

释放复制体的两种途径

• DOI: http://dx.10.1038/cdd.2017.75
• 发表时间: 2017
• 期刊: Cell Death & Differentiation
• 影响因子: 12.4
• 作者: D'Angiolella V

The Initiation of DNA Replication in Eukaryotes

真核生物中 DNA 复制的起始

• DOI: http://dx.10.1007/978-3-319-24696-3_17
• 发表时间: 2016
• 作者: De Piccoli G

Regulation of Unperturbed DNA Replication by Ubiquitylation.

通过泛素化调节不受干扰的 DNA 复制。

• DOI: http://dx.10.3390/genes6030451
• 发表时间: 2015
• 期刊: Genes
• 影响因子: 3.5
• 作者: Moreno SP

Termination of Eukaryotic Replication Forks.

真核复制叉的终止。

• DOI: http://dx.10.1007/978-981-10-6955-0_8
• 发表时间: 2017
• 期刊: Advances in experimental medicine and biology
• 作者: Gambus A