Defining A Role For DNA Phosphorylation In Maintaining Replication Fork Stability

Each of the cells in our body has approximately 2 meters of DNA that needs to be accurately copied every time a cell divides. Mistakes that occur during DNA copying are a major contributing factor towards the development of genetic diseases, cancer and early aging. DNA damage is one of the main causes that prevents cellular DNA being copied properly and can occur either through normal processes during cell growth or can be caused by exposure to radio/chemotherapy or environmental toxins. Excessive DNA damage is extremely toxic, and this forms the basis for why radio- or chemotherapy is used to kill tumour cells. However, cells have evolved complex mechanisms to recognise and repair DNA damage so that the DNA can be copied without any errors. When DNA is being copied, this is carried out by many proteins that work together as part of a big machine, called the replisome, to unzip, copy and rezip the entire DNA molecule. When the replisome finds damaged DNA, the copying process stops. If the DNA damage is not repaired properly and copying restarts, this can cause chromosomes to break and cells to die. However, when the replisome meets DNA damage, cells have developed an elegant process that pauses the copying process and allows the replisome to back track so that the DNA repair machinery can repair the damage. This is called replication fork (because as the DNA is unzipped and copied it looks like a 'fork') reversal. Replication fork reversal is a vital process for the repair and restart of DNA duplication, but represents a period of vulnerability. If the replisome and reversed replication fork are not stabilised and protected, the replisome can fall off and the newly copied DNA can be into pieces by proteins called nucleases. This can lead to the breaking or loss of chromosomes and increased mutations, all of which are known to contribute to human diseases. Whilst we understand a lot about how damaged DNA causes replication fork reversal and some of the proteins involved, there are still many unknowns. However, it is clear other proteins engaged in this process still remain to be identified. As such, it is imperative that when new proteins are found, their roles in controlling replication fork reversal are identified. This will give us vital knowledge about how healthy cells respond to DNA damage and what happens when this process goes wrong. This project will focus on understanding the function of a new protein called N4BP2 and how it controls replication fork reversal. N4BP2 is a unique protein in that it can modify DNA in two completely different ways: One is a chemical modification of the ends of DNA, called phosphorylation, and the other is to cut DNA when it forms unnatural shapes. This indicates that N4BP2 aids repair of damaged DNA undergoing duplication in two independent ways. Since there is no other protein in the cell similar to N4BP2, understanding how N4BP2 controls replication fork reversal could uncover a completely new mechanism with which damaged DNA is repaired before it is copied. We will use a combination of genetic and biochemical approaches to study N4BP2 function. First, we will identify what type of DNA damage N4BP2 recognises and what happens to the process of copying DNA when N4BP2 is lost. Second, we will identify what types of unusual DNA structures N4BP2 prefers to modify. Third, we will determine how N4BP2 controls individual proteins that make up the replisome at damaged forks. Combined, this approach will give us a good understanding of how N4BP2 regulates replication fork reversal to preserve DNA duplication and protect chromosomes from breaking. Lastly, these studies will complement a separate, but related study that is focused on determining the impact that loss of N4BP2 has on the development of embryos using a mouse model.