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

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.