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

所有细胞都以两步过程增殖，对于每个细胞，涉及基因组复制，然后是基因组分离，以提供两个活的子细胞。基因组复制通过DNA双螺旋的复制实现，其中在基因组分离过程之前，包含所有染色体的两条DNA链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。Fen 1是一种核酸酶--一种破坏将DNA碱基连接在一起的化学骨架的酶--它的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。核酸酶具有完全破坏DNA分子的潜力，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Fen 1的控制机制尚不清楚。了解Fen 1的调节方式很重要，因为在一系列癌症中-如结直肠癌-肿瘤演变成绝对依赖Fen 1功能，从而使Fen 1成为治疗开发的重要潜在靶点。Fen 1由一系列细胞开关控制，这些开关通过在Fen 1蛋白骨架上的离散位点处的一组复杂的调节酶对蛋白进行直接化学修饰而起作用。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，并建立基因改变的模型细胞系，这些细胞系表达Fen 1的版本，这些版本要么无法被修饰，要么蛋白质骨架被改变以永久模仿修饰。含有Fen 1改变形式的基因修饰细胞将用于高分辨率活细胞成像实验，以描述和理解Fen 1在DNA复制工厂背景下的分子行为。这些数据有望为开发新的分子实体提供未来的策略，这些分子实体将靶向Fen 1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.