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."