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.

细胞分裂是生命传播的基础。这涉及遗传信息的精确重复，这称为DNA复制。这个过程必须并且必须完全受到监管。任何未经修复的错误都可以改变细胞的行为和导致遗传疾病，癌症和衰老等疾病。令人着迷的是，在大多数情况下，细胞可以实现这一任务，即精确地毫无疑问地重复整个基因组。基因组重复对于快速分裂癌细胞也是必不可少的。这就是为什么许多抗癌疗法靶向DNA复制的原因，但到目前为止，这些疗法对癌细胞并不具体，并且具有明显的副作用。因此，至关重要的是，我们完全了解这一基本过程，以便我们可以制定更好，更具体的反癌策略。细胞已经开发出许多处理受损DNA以维持完整基因组的方法。最近几年揭示了称为泛素和SUMO在调节关键DNA修复蛋白中的小蛋白修饰剂的重要性。有强有力的证据表明，在复制未损坏的DNA期间，这些修饰也很重要。然而，对于它们靶向的蛋白质知之甚少。我项目的目的是调查泛素和相扑在未损坏的DNA复制过程中的作用。将使用两个生物系统来实现该项目。使用来自非洲爪蛙（Xenopus laevis）的卵中的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成型络合物，因此可以在试管中支持完整的DNA复制。由于此过程的许多方面在所研究的所有真核生物中都非常相似，因此在较简单的鸡蛋提取物模型系统中鉴定的机制在人类细胞中通常也是如此。一旦已经鉴定出了新的修饰并在爪蟾卵提取物中进行了基本机理研究，我想研究类似机制在人类细胞中是否也功能。尤其是我有兴趣研究泛素在DNA复制终止阶段的作用，如我的初步数据所暗示的那样。当两个DNA复制叉来自染色体融合的相对位点时，复制终止就会发生。这些结构的有效且完美的分辨率对于维持基因组完整性至关重要。在每个终止阶段必须解决人类细胞中成千上万的复制叉 - 因此，至关重要的是，我们创建了研究此过程的工具并确定其对肿瘤发生的输入。但是，这一DNA复制的阶段也非常了解，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及其破坏后果的机制。以类似的方式，我将研究阻断sumoylation对DNA复制不同方面的影响。我还希望利用Xenopus系统中DNA复制的生化分析的简单性，并对蛋白质进行系统分析，对蛋白质进行系统分析，并在DNA复制过程中对复制DNA关联并进行了修改。然后，我将选择最有趣的方法，并表征这些修改的类型和站点。我的最终目的是确定确定的修饰的功能及其在DNA复制和癌症发育过程中的重要性。指定在DNA复制过程中泛素和SUMO修改的作用，将扩大我们对这一过程的理解。 DNA复制和泛素系统均由许多当前的抗癌化学疗法靶向。因此，在这两者之间揭示新的串扰途径可能表明化学治疗剂的新靶标或组合可能会揭示产生对癌细胞特别致命的DNA损伤的新方法。

项目成果

Two paths to let the replisome go.

• DOI: 10.1038/cdd.2017.75
• 发表时间: 2017-07
• 期刊: Cell death and differentiation
• 影响因子: 12.4
• 作者: D'Angiolella V;Guardavaccaro D
• 通讯作者: Guardavaccaro D

Mitotic replisome disassembly in vertebrates

• DOI: 10.1101/418368
• 发表时间: 2018-09
• 期刊: bioRxiv
• 作者: S. Moreno;Rebecca M Jones;Divyasree Poovathumkadavil;Agnieszka Gambus

Regulation of Unperturbed DNA Replication by Ubiquitylation.

• DOI: 10.3390/genes6030451
• 发表时间: 2015-06-25
• 期刊: Genes
• 影响因子: 3.5
• 作者: Moreno SP;Gambus A
• 通讯作者: Gambus A

The Initiation of DNA Replication in Eukaryotes

真核生物中 DNA 复制的起始

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

Termination of DNA replication forks: "Breaking up is hard to do".

• DOI: 10.1080/19491034.2015.1035843
• 发表时间: 2015
• 期刊: Nucleus (Austin, Tex.)
• 作者: Bailey R;Priego Moreno S;Gambus A
• 通讯作者: Gambus A