Novel Ubiquitin Ligases and DUBs in DNA Replication and Damage

Protein ubiquitylation provides a versatile protein regulation system influencing almost every aspect of cell biology. Within the last few years we have significantly contributed to the current understanding of the role ubiquitylation plays during the process of DNA replication, specifically during the termination of replication forks. The overarching aim of the PhD project is to identify and characterise interesting and understudied ubiquitin ligases and deubiquitylating enzymes (DUBs) that are potentially involved in the termination of DNA replication. Through four defined experimental objectives we will determine the function of the selected enzymes during DNA replication, replication stress and damage response, and assess them as potential therapeutic targets. Objective 1: to characterise when the studied enzymes interact with chromatin and their potential substrates. Objective 2: Understand the function and mechanism of action of studied enzymes during DNA replication and damage. Objective 3: Determine phenotype of down-regulation of these enzymes in human cells and its link with DNA replication/damage. Objective 4: Investigate differential requirements of the studied enzymes in normal and cancerous cell lines. In order to accomplish these aims, we will utilise a combination of biochemical and cell biology approaches using cell free Xenpus laevis egg extract and human immortalised cell lines. It is thought that the proposed research has the potential to expand our knowledge of the importance of ubiquitylation during DNA replication and damage, in particular, of the underlying mechanisms and regulatory processes surrounding the termination of replication forks.