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

所有细胞的增殖分两步进行，每个细胞都需要进行基因组复制，然后进行基因组分离，从而产生两个有活力的子细胞。基因组复制是通过复制DNA双螺旋实现的，其中组成所有染色体的两条DNA链在基因组分离过程之前复制一次，并且只复制一次。DNA复制是在复制工厂进行的，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--一种打破连接DNA碱基的化学主干的酶--它负责修剪新合成的DNA链，以确保在基因组复制过程中不会丢失基因组信息。核酸酶有可能完全摧毁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的调控机制尚不清楚。了解Fen1的调控方式很重要，因为在一系列癌症中--如结直肠癌--肿瘤演变成完全依赖Fen1功能，从而使Fen1成为治疗开发的重要潜在靶点。Fen1是由一系列细胞开关控制的，这些开关通过位于Fen1蛋白骨架上离散位置的一组复杂的调控酶直接对蛋白质进行化学修饰。该项目的目的是使用质谱学确定这些化学修饰的准确位置和性质，并建立基因改变的模型细胞系，这些细胞系表达无法修改的Fen1版本，或者其中蛋白质骨架被改变以永久模拟修改。含有改变形式的Fen1的转基因细胞将被用于高分辨率活细胞成像实验，以在DNA复制工厂的背景下描述和理解Fen1的分子行为。这些数据有望为未来开发新的分子实体提供信息，这些新的分子实体将靶向Fen1以获得治疗益处。

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.