In vivo analysis of context-specific post translational control of Fen1: its role in genome stability and therapeutic target potential

Fen1 上下文特异性翻译后控制的体内分析：其在基因组稳定性和治疗靶点潜力中的作用

• 批准号: 1991278
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1991278
• 关键词: vivo; analysis; context; specific; post

All cells proliferate in a two-step process that for each cell involves genome duplication followed by genome segregation to deliver two viable daughter cells. Genome duplication is achieved via replication of the DNA double helix, wherein both DNA strands comprising all chromosomes are copied once, and only once, before the process of genome segregation. DNA replication is undertaken in replication factories -distinct regions in a cell nucleus dedicated to the production of newly synthesised chromosomes. Fen1 is a nuclease - an enzyme that breaks the chemical backbone linking DNA bases together - that acts to trim newly synthesised DNA strands to ensure that there is no loss of genomic information during the process of genome duplication. Nucleases have the potential to destroy DNA molecules completely, and cells have evolved complex control systems to ensure that they are highly regulated. The mechanisms by which Fen1 inside the cell is controlled are not understood. Understanding the way Fen1 is regulated is important because in a range of cancers - such as colorectal cancer - tumours evolve to become absolutely dependent on Fen1 function, thus making Fen1 an important potential target for therapeutic development. Fen1 is controlled by a range of cellular switches that work by direct chemical modification of the protein via complex set of regulatory enzymes at discrete sites on the protein backbone of Fen1. The aim of this project is to identify the precise location and nature of these chemical modifications using mass spectrometry, and to build genetically altered model cell lines that express versions of Fen1 that are either unable to be modified, or where the protein backbone is altered to permanent mimic a modification. Genetically modified cells containing altered forms of Fen1 are to be utilised in high-resolution live-cell imaging experiments to describe and understand the molecular behaviour of Fen1 in the context of a DNA replication factory. These data are expected to inform future strategies for the development of novel molecular entities that would target Fen1 for therapeutic benefit.

所有细胞都通过两步过程增殖，每个细胞都涉及基因组复制，然后进行基因组分离，以产生两个可行的子细胞。基因组复制是通过DNA双螺旋的复制实现的，其中构成所有染色体的两条DNA链在基因组分离过程之前被复制一次，且仅复制一次。 DNA 复制是在复制工厂中进行的，复制工厂是细胞核中专门用于生产新合成染色体的不同区域。 Fen1 是一种核酸酶 - 一种破坏将 DNA 碱基连接在一起的化学主链的酶 - 其作用是修剪新合成的 DNA 链，以确保在基因组复制过程中不会丢失基因组信息。核酸酶有可能完全破坏 DNA 分子，细胞已经进化出复杂的控制系统以确保它们受到高度调控。细胞内 Fen1 的控制机制尚不清楚。了解 Fen1 的调节方式非常重要，因为在一系列癌症（例如结直肠癌）中，肿瘤会进化为完全依赖于 Fen1 功能，从而使 Fen1 成为治疗开发的重要潜在靶点。 Fen1 受一系列细胞开关控制，这些细胞开关通过 Fen1 蛋白质骨架上离散位点上的一组复杂的调节酶对蛋白质进行直接化学修饰来发挥作用。该项目的目的是使用质谱法确定这些化学修饰的精确位置和性质，并构建基因改变的模型细胞系，这些细胞系表达 Fen1 的版本，这些版本要么无法被修饰，要么蛋白质主链被改变以永久模拟修饰。含有改变形式的 Fen1 的转基因细胞将用于高分辨率活细胞成像实验，以描述和理解 DNA 复制工厂中 Fen1 的分子行为。这些数据预计将为未来开发以 Fen1 为靶点以获得治疗益处的新型分子实体的策略提供信息。