Genomic Health & Safety: Does Elg1 maintain genome stability by resetting chromatin factors used in DNA replication and repair?

The preservation of correct and accurate information is critical in almost all aspects of life. The information that enables life is encoded in DNA, which is folded up with proteins inside cells to form chromosomes. The failure to maintain chromosomes leads to the loss of or rewriting of important information, which can cause cancer and other diseases. The protein called Elg1 is a cellular factor that is very important in maintaining chromosomes. Defects in this one protein cause tumors in humans and mice, but we do not know why. The aim of this work is to understand how the Elg1 protein maintains chromosome stability so that we may exploit these findings to develop new cancer therapies.

Whenever a cell divides to produce two new cells, the genetic information in the chromosomes must be duplicated precisely. This duplication process is called DNA replication. In most cases, cells achieve precise DNA replication without any mistakes. Hundreds of proteins in cells contribute to such precise DNA replication. If such 'chromosome stability' proteins cannot carry out their proper functions, cell cannot duplicate genetic information precisely, resulting in loss of or rewriting of important information, and leading eventually to cancer, genetic disorders, and ageing. A protein called Elg1 is one of these chromosome stability proteins. Recently I discovered that the molecular function of Elg1 is removal of a sliding clamp called PCNA from DNA during DNA replication. PCNA is ring-shaped and encircles DNA. PCNA stabilises the machinery that copies DNA, and additionally the PCNA ring acts like a tool-belt, recruiting many other collaborating proteins that are important for chromosome maintenance and precise DNA replication. During DNA replication, the PCNA tool-belt is repeatedly loaded on DNA, and repeatedly removed from DNA by Elg1 after each section of the DNA replication task is complete. In cells lacking Elg1, the PCNA tool-belt and its tools (i.e. its collaborating proteins) accumulate on chromosomes since the PCNA tool-belt is not removed even after completion of each new DNA section. This abnormal accumulation of the PCNA tool-belt resembles a construction worker with ten tool-belts around his body, each containing several unnecessary tools. Workers carrying ten tool-belts and lots of unnecessary tools may be not able to move their bodies flexibly, respond effectively to unexpected events, or use tools efficiently-and so will be more liable to make mistakes. In chromosome stablity, even occasional mistakes may cause catastrophe. My hypothesis is that the aberrant accumulation on DNA of the PCNA tool-belt and its associated tools causes loss of or rewriting of genetic information during chromosome duplication, ultimately resulting in cancer.

I aim to understand why PCNA removal by Elg1 is important for chromosome maintenance, and whether its collaborating proteins are involved. First, by manipulating the PCNA tool-belt, I will test whether unwanted accumulation of the PCNA tool-belt interferes directly with chromosome maintenance. Next I will test whether the accumulation of particular unnecessary tools (i.e. unwanted retention of the collaborating proteins) contributes to chromosome instability. These experiments will be carried out using the baker's yeast system, which allows sophisticated molecular genetic approaches to be used for careful dissection of the chromosome stability machinery. In the third part, I am keen to extend this investigation to test the role of Elg1 in human cells, since loss of Elg1 is directly implicated in mammalian tumors. Since this project studies the mechanism of action of gene that is associated with cancer, this work holds long-term potential for cancer therapy.

Maintenance of genome stability is critical to avert diseases and cancer. Loss of the genome maintenance factor Elg1 leads to genome instability and results in tumorigenesis, but we do not know why. The ultimate aim of this work is to understand how Elg1 maintains genome stability.

Recently I discovered that the Elg1 Replication Factor C-like complex functions to unload the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp during DNA replication. PCNA is a central protein in DNA replication and repair, and is often used as marker of cell proliferation and malignancy. PCNA acts as a sliding scaffold for many replication and repair proteins. In the absence of Elg1, PCNA and its interacting partners accumulate on chromatin. I hypothesise that the abnormal accumulation of PCNA and its interacting partners on chromatin leads to the genome instability observed in cells lacking Elg1.

To test this hypothesis, I will manipulate PCNA in budding yeast and test whether abnormal accumulation of PCNA on chromatin is responsible for genome instability in cells lacking Elg1. I will next test whether the accumulation of specific PCNA-interacting partners on chromatin contributes to genome instability, and also investigate the function of PCNA comprehensively both in DNA replication and repair. The powerful genetics approaches possible in yeast allow the sophisticated dissection of chromosome stability pathways. Lastly I will extend my investigation to human cells, to test whether similar mechanisms operate. Addressing how Elg1 maintains human chromosome stability is an important step since loss of Elg1 is directly implicated in mammalian tumors.

This research will substantially advance our understanding of how cells ensure genome stability by illuminating the role of the genome maintenance factor Elg1 in PCNA regulation. Since this project studies the mechanism of action of gene that is associated with cancer, this work holds long-term potential for cancer therapy.

My research will lead to a greater understanding of how cells maintain stability of their genomes as they multiply. Many human diseases and cancer are caused by the derailment of the normal biological processes that are responsible for maintaining genome stability. Therefore, understanding the mechanisms of genome maintenance and how the loss of genome stability causes disease will inform thinking on how to treat such diseases, which include DNA repair syndromes (e.g. Xeroderma pigmentosum, Cockayne's syndrome, ataxia telangiectasia), repeat expansion disorders (e.g. Huntingdon's disease, Friedrich's ataxia) and numerous other diseases of the genome including cancer. As such, this work may also reveal new targets for the development of future therapeutic agents. This research will have both immediate and long-term impacts.

Who will benefit from this research?
The main and immediate benefit of this work will be to the contribution of knowledge and understanding of the mechanisms that maintain chromosomes in dividing cells. The academic impact of this work is described in the Academic Beneficiaries section. More immediately, researchers working on this project will benefit from this work. In the longer-term, beneficiaries will include pharmaceutical companies, cancer charities, individuals suffering from cancer and other genome instability disorders, clinicians treating these patients, and the wider public.

How will they benefit from this research?
1. Researchers working on this project
The PI and a research assistant working on this project will receive training in experimental skills, designing studies, and communicating the importance of their research. This work therefore will contribute to maintaining a highly-skilled workforce in the UK.

2. Pharmaceutical companies and cancer charities
The process of DNA replication is a proven target of anticancer therapies. This research investigates the functions of DNA replication factors. Advances in the understanding of DNA replication will help the understanding of how these therapies work and may assist in the design of new diagnostic strategies and new therapeutic drugs.

3. Individuals suffering from cancer and other genome instability disorders and clinicians treating them.
My proposed work centres around Elg1 and PCNA (Proliferating Cell Nuclear Antigen) which are important factors that maintain and support genome stability, DNA replication, repair, and recombination. Defects in these factors are related to cancer and diseases. In the long term, understanding the mechanism by which dysfunction of these factors results in cancer has tremendous potential to inform the development of new cancer therapies.

4. The Wider public
Public understanding of science is a societal benefit, contributing to an educated workforce, and to general health. This project will reveal how loss of a gene causes genome instability, ultimately resulting in cancer. The research outcome therefore can be applied for genetic diagnosis of potential tumorigenesis.