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。这一过程必须而且已经得到精确的管制。任何随后没有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无误地复制整个基因组的任务。基因组复制对于快速分裂的癌细胞也是必不可少的。这就是为什么许多抗癌疗法以DNA复制为目标，但迄今为止这些疗法并不是针对癌细胞的，并且具有显着的副作用。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策略。细胞已经开发出许多处理受损DNA的方法，以保持完整的基因组。最近几年，人们发现了称为泛素和SUMO的小蛋白修饰剂在调节关键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，人们对它们所靶向的蛋白质知之甚少。我的项目的目的是研究泛蛋白和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这个项目。使用非洲爪蟾（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蛋白质的预形成复合物，因此可以支持试管中完整的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高度相似，因此在更简单的卵提取物模型系统中鉴定的机制在人类细胞中也是最常见的。一旦新的修饰已经确定，并已在非洲爪蟾卵提取物进行了基本的机制研究，我想调查是否类似的机制功能也在人类细胞。特别是，我感兴趣的是调查的作用，泛素在终止阶段的DNA复制，建议由我的初步数据。当两个来自染色体相对位点的DNA复制叉融合在一起时，复制终止发生。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。在人类细胞中有成千上万的复制叉，必须在每个终止阶段解决-因此，我们创造工具来研究这个过程并确定其对肿瘤发生的输入至关重要。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破坏的后果。以类似的方式，我将研究阻断sumoylation对DNA复制不同方面的影响，我还想利用非洲爪蟾系统中DNA复制的生物化学分析的简单性，对与DNA复制相关的蛋白质和在DNA复制过程中修饰的蛋白质进行系统分析。然后，我将选择最有趣的，并描述这些修改的类型和位置。本论文的最终目的是确定这些修饰的功能以及它们在DNA复制和癌症发生过程中的重要性，确定泛素和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目前许多抗癌化疗的目标。因此，揭示这两者之间的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异性致命的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

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

DNA复制叉的终止：“分手很难做到”。

• 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

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