Roles of ubiquitin and SUMO during chromosomal DNA replication.

Cell division is the basis for the propagation of life. This involves the precise duplication of genetic information, which is called DNA replication. This process must be, and is, precisely regulated. Any mistakes that are not subsequently repaired can change the way the cell behaves and result in conditions such as genetic diseases, cancer and ageing. It is fascinating that in most cases cells can achieve this task of duplicating the whole genome precisely once and without mistakes. Genome duplication is also essential for fast dividing cancer cells. It is why many anti-cancer therapies target DNA replication, but these are so far not specific for cancer cells and have significant side-effects. It is therefore crucial that we fully understand this fundamental process so that we can develop better and more specific anti-cancer strategies.

Cells have developed many ways of dealing with damaged DNA to maintain an intact genome. Last few years brought to light the importance of small-protein modifiers called ubiquitin and SUMO in regulating key DNA repair proteins. There is strong evidence to suggest that these modifications are also important during the replication of undamaged DNA. However little is known about the proteins they are targeting. The aim of my project is to investigate the role of ubiquitin and SUMO during replication of undamaged DNA.

Two biological systems will be used to fulfill this project. The cell-free system using extracts from the eggs of the African clawed frog (Xenopus laevis) contains pre-formed complexes of most proteins required for cell cycle progression and so can support a complete round of DNA replication in a test tube. As many aspects of this process are highly similar in all eukaryotic organisms studied, the mechanisms identified in simpler egg extract model system are most often true also in human cells. Once novel modifications have been identified and basic mechanistic studies have been performed in Xenopus egg extract I would like to investigate whether analogous mechanism function also in human cells.

In particular, I am interested in investigating the role of ubiquitin during the termination stage of DNA replication, as suggested by my preliminary data. Replication termination occurs when two DNA replication forks coming from the opposite sites of the chromosome fuse together. The efficient and faultless resolution of these structures is crucial for maintaining the genome integrity. There are thousands of replication forks in human cell which have to be resolved during each termination phase - it is crucial therefore that we create tools to study this process and determine its input towards tumorgenesis. However, this stage of DNA replication is also very poorly understood and therefore a very exciting research area. I will study the mechanism by which ubiquitylation regulates this process and the consequences of its disruption. In an analogous way, I will examine the effects of blocking sumoylation on different aspects of DNA replication.

I would also like to take advantage of the simplicity of biochemical analysis of DNA replication in Xenopus system and perform a systematic analysis of proteins associating with replicating DNA and modified during DNA replication. I will then choose the most interesting ones and characterize the type and site of these modifications. My final aim is to determine the function of the identified modifications and their importance in process of DNA replication and cancer development.

Defining the role of ubiquitin and SUMO modifications during DNA replication will widen our understanding of this process. Both DNA replication and the ubiquitin system are targeted by many current anti-cancer chemotherapies. Unveiling new crosstalk pathways between these two may therefore suggest novel targets or combinations of chemotherapeutic agents as it may reveal new ways of creating DNA damage specifically lethal to cancer cells.

Post-translational modification of proteins by members of the ubiquitin family: specifically ubiquitin and SUMO, is essential in DNA damage response pathways. However their role during unperturbed DNA replication not well understood.
The initiation stage of DNA replication depends on ubiquitin-mediated proteolysis. However, a growing body of evidence suggest that the further stages of DNA replication are also regulated by these modifications. The importance of ubiquitin/SUMO ligases during unperturbed DNA replication has been shown, but very little is known about their substrates. My aim is therefore to use my experience in studying protein complexes assembled during DNA replication to investigate their regulation by ubiquitin and SUMO.
For the first part of my project I will use the cell-free Xenopus egg extract system, which provides a powerful system for the biochemical analysis of eukaryotic chromosomal replication. I will identify proteins ubiquitylated and sumoylated during DNA replication by mass spectrometry analysis. I will then select most interesting candidates and map the modifications sites and their function during DNA replication.
My preliminary data suggest an important role for polyubiquitylation in the disassembly of the replisome upon termination of DNA replication forks. These findings are of the highest interest as at present we have little knowledge of this process. I will, therefore, study the mechanism and the consequences of this defect. I will also investigate the effects of blocking sumoylation on replisome formation, regulation and disassembly.
Once novel ubiquitylated and sumoylated proteins have been identified and basic mechanistic studies have been performed using Xenopus egg extracts I will select a few most interesting substrates and study their modifications in human cell lines.

My research will have an academic impact: enhancing the knowledge and worldwide academic advancement. I will also train future researchers.

The research I propose will lead to a greater understanding of the fundamental process of eukaryotic DNA replication. Problems arising during DNA replication are the main source of genomic instabilities that can lead to mutations and multiple diseases including cancer. The better understanding of this important process and the ways in which it can malfunction will therefore allow better understanding of the development of these diseases. Moreover, the process of DNA replication is also one of the main targets of anticancer therapies. Therefore advances in the understanding of the functioning of this process will help understanding how these therapies work and the design of more specific therapeutic agents. Altogether, in the long term my research will enhance the scientific knowledge leading to design of novel, better drugs and drugs combinations. The proposed research is therefore a basic research that will contribute to improved health and treatment outcomes in the long term.

Public beneficiaries of this research will include cancer patients, patients suffering from genetic disorders caused by mutations in replication factors, the NHS, cancer charities and biotechnology companies.

1. Cancer patients
Better treatments for cancer are of general interest among the public but especially among the cancer sufferers. To achieve this goal we need to better understand the ways cancer develops, the mechanisms by which current cancer therapies work to improve them and widen our knowledge of processes essential for cancer phenotype which can be targeted by new drugs. Many cancer patients are treated with radiotherapy and chemotherapy targeting DNA replication which although effective are dose-limited and cause many side effects. It is therefore of high interest to develop new targets affecting the essential for cancer development process of DNA replication, which will be as efficient as current drugs but much more specific for cancer cells.

2. Patients suffering from genetic disorders caused by mutations in replication factors (such as Rothmund-Thomson, Rapadilino syndromes caused by mutation in RecQ4)
As my research will increase our understanding of regulation of process of DNA replication it may increase our understanding of development of these disorders and may suggest novel ways to help patients with conditions caused by mutations in replication factors.

3. The NHS, cancer charities, pharmaceutical companies
All these organisations seek development of better, more specific anti-cancer drugs. The proposed research will be therefore of benefit for all of them.

Finally, through public engagement I would like to contribute to increase of public awareness and understanding of science and share my enthusiasm towards science with public.