A multi-tool for chromosome maintenance: how does Elg1 guard genome stability?

All cells contain a copy of the organism's DNA, wrapped up into chromosomes and forming the genome, which acts as the cellular operating system. For proper cell function it is crucial that the genome is correctly maintained, and critical that perfect copies of the chromosomes are passed to new cells during development or tissue regeneration.

Cells have a remarkable molecular 'toolkit' containing all the implements needed for different aspects of chromosome maintenance--tools for repairing damaged DNA, for copying the DNA, for packaging it properly into chromosomes, and for segregating chromosome copies to daughter cells. For example, a component called 'PCNA' has a central function during DNA replication. PCNA is ring-shaped and encircles replicating DNA, acting like a sophisticated washer or 'hex nut' to coordinate the DNA-copying machinery. The PCNA replication coordinator is loaded onto DNA by a spanner-like tool called Replication Factor C. The tool that unloads PCNA once replication is completed has not yet been identified.

Our recent discoveries suggest that PCNA is unloaded by a component called Elg1, which acts as a multi-functional tool during DNA replication. Elg1 is clearly important: it exists in organisms as distantly related as yeast and human, and its loss leads to problems such as chromosome mutation, loss, and rearrangement--defects which in humans cause neurological syndromes (e.g. Huntington's disease), birth defects (e.g. Down's syndrome), and diseases like cancer. Despite its importance, until now we have had limited understanding of the molecular function of Elg1. This project represents a golden opportunity to understand how Elg1 maintains genome stability.

This work will test our proposal that Elg1 acts first as a 'spanner' to unload the ring-shaped PCNA component from newly-replicated DNA, and then as an 'adaptor' that recruits packaging machinery to assemble newly replicated DNA correctly into chromosomes. The suggestion that Elg1 unloads PCNA once replication is complete is consistent with the fact that Elg1 is structurally similar to Replication Factor C, the molecular spanner that loads PCNA as replication begins.

Within cells, DNA is packaged around miniscule bobbin-like structures called nucleosomes. DNA must be unwound for replication to take place and, afterwards, the replicated DNA must be correctly re-wound around nucleosomes. Failure to re-package DNA properly causes problems in delivering the new chromosomes to daughter cells (as a parcel whose string isn't tied will fall apart in the post). Cells contain a series of DNA 'packaging factors' responsible for re-winding of DNA following replication. We have discovered that Elg1 interacts with one of these packaging factors, suggesting a further role for Elg1 is to ensure DNA is properly re-wound around nucleosomes following replication. The proposed work will investigate this possibility.

Our initial experiments will examine the role of Elg1 using baker's yeast. The sophisticated experiments possible using yeast allow us to test our central hypotheses for Elg1 function. Of course we wish to confirm that Elg1 in higher organisms acts in the same way. We will examine the function of vertebrate Elg1 using a replication system derived from frog eggs, which allows incisive analysis of the DNA replication machinery. The replication machinery is virtually identical in frog and human cells, so our findings in frog can be extended to understand how Elg1 acts in human cells.

Overall, this work will elucidate the role of Elg1 as a multifunctional genome maintenance tool. As an added benefit, our work will investigate why the problems resulting from loss of Elg1 cause chromosome instability and ultimately lead to cancer.

This work addresses BBSRC strategic research priorities Basic Research for Health, Biology of Ageing, and Extension of International Collaboration.

Accurate chromosome maintenance is critical to avert diseases caused by genome instability. The aim of this work is to elucidate the role in DNA replication of the genome stability component Elg1. It is important that we understand the basic biological function of Elg1 in maintaining chromosomes, since its loss or mutation causes complex problems with genome stability that lead inexorably to cancer.

Elg1 is the major subunit of a 'Replication Factor C-Like (RLC) Complex', similar in structure to Replication Factor C which loads the ring-shaped complex PCNA (Proliferating Cell Nuclear Antigen) at replication forks. Our preliminary results suggest that Elg1-RLC also acts on PCNA. PCNA encircles DNA, acting as a clamp for DNA polymerases and as a recruitment platform for proteins that mediate replication-associated processe--including chromatin assembly, the DNA re-packaging step which is critical for ensuring faithful transmission of genomic information.

Based on our pilot results, we hypothesise that the central function of Elg1-RLC is to unload PCNA from replication forks, and the first part of the work will test this possibility in yeast. The second part of the project will test our proposal that Elg1-RLC recruits the histone chaperone Rtt106 to replication forks, to ensure that PCNA unloading is coupled to chromatin re-assembly. While exploring linked hypotheses, these first and second parts can be pursued independent of each other's results. In the third section, we will exploit the biochemically amenable Xenopus in vitro replication system, to test whether vertebrate Elg1-RLC also mediates PCNA unloading and chromatin assembly. An additional aim will be to investigate how loss of Elg1 function causes chromosome instability.

Overall, this research will substantially advance our understanding of how cells ensure genome stability by illuminating the role of the central conserved chromosome maintenance component Elg1.

As basic biology this research will have some immediate and many potential long-term impacts. Since human disease is caused by derailment of normal biological processes, understanding basic biological mechanisms is of paramount importance. Beneficiaries will include individuals suffering from cancer and other genome instability disorders, clinicians treating these patients, the elderly popoulation, pharmaceutical companies, charities, and the wider public, as well as the UK and international researchers involved with this project.

A major route to long-term impact arises from improved understanding of the mechanisms that maintain chromosomes, and of how genome stability is adversely affected by defects in these mechanisms. For continuing life every organism depends on correct maintenance and copying of its genome, which forms its genetic blueprint. Loss of Elg1 function has notably diverse (and always delerious) consequences for genomes. Inadequate Elg1 function causes cancer, and this work will reveal how such oncogenesis relates to defects of cells lacking Elg1 -- including their acute DNA damage sensitivity (with impact for drug treatments involving DNA damage), reduced DNA replication efficiency (impacting on understanding cancer and use of proliferation biomarkers), compromised telomere function (with impact for understanding ageing), and elevated genome rearrangements. Understanding how loss of Elg1 causes these diverse problems will be critical for understanding how cells maintain their genomes in the face of environmental and biological challenges.

As such, this research ultimately holds the potential to impact virtually everybody, from embryos in utero (at risk of miscarriage due to congenital defects), through to the very elderly (whose genomes are frail due to damage accumulation and telomere erosion), and of course including normal individuals (exposed daily to carcinogens like sunlight) and patients with genome instability disorders (that predispose to carcinogenesis).

Understanding the role of the Elg1 and other Replication Factor C-like Complexes therefore has substantial long-term potential to improve treatment of individuals affected by any of these genomic problems, through the development by pharmaceutical companies of improved and novel drugs, and through better understanding by clinicians and clinical researchers of how existing drugs work and how patient responses to them may be controlled.

More immediately, this work will have important impact on the wider public (through our planned scientific outreach activities), and on charities and fund-raisers involved with research of genome instability syndromes and patient support groups.

Additional, immediate impact arises from the international collaborations that this work involves, which will raise and maintain the profile of UK research through our interactions with researchers in Japan and the USA. In the medium-term these collaborations are likely to lead to further collaborative grant applications to UK and international funders, providing national and international economic impact.

There will moreover be substantial important, immediate impact from the training of researchers working on this project, who will be well-placed and highly competent to design and pursue studies of the highest future significance, and to communicate the importance of their research--to other academics, to students in training, and to the public. This work will therefore also contribute to maintaining a workforce with a high level of scientific literacy, in the long-term ensuring continuing competitiveness of the UK economy.