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 cut 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.
This project will define how DNA damage is processed to maintain normal DNA replication and protect genomic integrity. This work will help us understand the mechanisms controlling how tumour cells respond to chemotherapy and how these processes are subverted to promote the development of resistance.
Grant Stewart, Professor of Cancer Genetics, University of Birmingham
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 discovered. As such, it is imperative that when new proteins are found, their roles in controlling replication fork reversal are characterised. 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 that controls replication fork reversal by modifying DNA ends. We will use a combination of genetic and biochemical approaches to study: (1) how this new fork reversal protein recognises damaged replication forks and what happens to the process of copying DNA when it is lost, (2) what types of DNA substrate are recognised by the two enzymatic activities of this new fork reversal protein and (3) whether this new fork reversal protein alters the constituents of the replisome at damaged forks. Combined, this approach will give us a detailed understanding of how this previously undiscovered protein regulates replication fork stability 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 this new fork reversal protein has on the development of embryos using a knockout mouse model.
BBSRC funding this project means a great deal to the Stewart and Smerdon laboratories, especially during this time when research funding is scarce and what funding is available is extremely competitive. We believe that the outputs from this project will not only provide us with insight into how fundamental processes like DNA replication are regulated but they could also have translational implications for understanding how tumour cells respond to chemotherapy.
