DNA damage triggers disruption of telomeric silencing and Mec1p-dependent relocation of Sir3p.

In eukaryotic cells, surveillance mechanisms detect and respond to DNA damage by triggering cell-cycle arrest and inducing the expression of DNA-repair genes [1]. In budding yeast, a single DNA double-strand break (DSB) is sufficient to trigger cell-cycle arrest [2]. One highly conserved pathway for repairing DNA DSBs is DNA non-homologous end-joining (NHEJ), which depends on the DNA end-binding protein Ku [3]. NHEJ also requires the SIR2, SIR3 and SIR4 gene products [4] [5], which are responsible for silencing at telomeres and the mating-type loci [6]. Because of the link between NHEJ and the Sir proteins, we investigated whether DNA damage influences telomeric silencing. We found that DNA damage triggers the reversible loss of telomeric silencing and relocation of Sir3p from telomeres. Complete Sir3p relocation was triggered by a single DNA DSB, suggesting that the singal is amplified. Consistent with this idea, Sir3p relocation depended on the DNA damage-signalling components Ddc1p and Mec1p. Thus, signalling of DNA damage may release Sir3p from telomeres and permit its subsequent association with other nuclear subdomains to regulate transcription, participate in DNA repair and/or enhance genomic stability by other mechanisms.

Localisation and interaction of the protein components of the yeast 2 mu circle plasmid partitioning system suggest a mechanism for plasmid inheritance.

Replicating plasmids are highly unstable in yeast, because they are retained in mother cells. The 2 mu circle plasmid overcomes this maternal inheritance bias by using a partitioning system that involves the plasmid encoded proteins Rep1p and Rep2p, and the cis-acting locus STB. It is thus widely exploited as a cloning vehicle in yeast. However, little is known about the cellular or molecular mechanisms by which effective partitioning is achieved, and models of both free diffusion and plasmid localisation have been proposed. Here we show that Rep1p and Rep2p proteins interact to form homo- and hetero-complexes in vitro. In vivo, Rep1p and Rep2p are shown to be nuclear proteins, exhibiting sub-nuclear concentration in distinct foci. The number of foci appears constant regardless of plasmid copy number and cell ploidy level. Before cell division, the number of foci increases, and we observe approximately equal allocation of foci to mother and daughter cell nuclei. We show that whereas Rep2p expressed alone is found exclusively in the nucleus, Rep1p requires the presence of Rep2p for effective nuclear localisation. High levels of 2 mu plasmid induce a multiple-budded elongated cell phenotype, which we show can be phenocopied by overexpression of both REP1 and REP2 together but not alone. Taken together, these results suggest that Rep1p and Rep2p interact in vivo, and occupy defined nuclear sites that are allocated to both mother and daughter nuclei during division. We propose a model for 2 mum plasmid partitioning based on these results, involving the association of plasmid DNA with specific, segregated subnuclear sites.

Inhibition of oligodendrocyte precursor motility by oligodendrocyte processes: implications for transplantation-based approaches to multiple sclerosis.

Transplantation of oligodendrocyte precursor cells represents a promising approach to the treatment of the chronic demyelinated lesions of multiple sclerosis. In view of the multi-focal nature of the disease it will be necessary for the transplanted oligodendrocyte precursor cells to migrate through normal white matter between lesions. Work in other systems has shown that differentiated oligodendrocytes within white matter express molecules inhibitory for axon outgrowth. In light of this we have examined the effect of oligodendrocytes on the migration of oligodendrocyte precursors in vitro using time lapse video microscopy. We find that oligodendrocytes induce collapse and loss of motility in oligodendrocyte precursor processes, with this effect being lost as oligodendrocytes undergo programmed cell death. We conclude that the inhibitory factors present on differentiated oligodendrocytes may prevent effective migration between lesion in vivo, and that strategies to overcome this inhibition may be required for successful repair.

Expression and function of thrombospondin-1 in myelinating glial cells of the central nervous system.

The thrombospondin (TSP) family of extracellular matrix glycoproteins are widely expressed in the developing and adult central nervous system although their function remains poorly defined. We have used cell culture techniques to analyse the expression and function of TSPs in glial cells derived from myelinated regions of the central nervous system. These experiments show that TSP-1 mRNA, but not TSP-2 or TSP-3 mRNA, is expressed by astrocytes from these regions. TSP-1 mRNA levels in astrocytes are under the regulation of growth factors, being increased by TGFbeta1 and decreased by bFGF. Oligodendrocyte precursors do not express TSP-1, TSP-2, or TSP-3 mRNA. Migration of oligodendrocyte precursor cells is stimulated by TSP-1 substrates as measured either by time-lapse microscopy or using a microchemotaxis chamber assay. Taken together, these results suggest that the extracellular matrix molecule TSP-1 plays a role in normal central nervous system development by contributing to the regulation of oligodendrocyte precursor migration.