The stable maintenance of the 2m circle plasmid depends on its ability to overcome intrinsic maternal inheritance bias, which in yeast normally results in the failure to transmit DNA molecules efficiently to daughter cells. In addition to the plasmid proteins Rep1 and Rep2 acting on the plasmid DNA locus STB, it is likely that other chromosomally encoded yeast proteins are required. We have isolated mutants of yeast unable to maintain 2m and found that RSC2 is essential for 2m to overcome maternal inheritance bias. Rsc2 is part of a multisubunit RSC chromatin remodeling complex, and we show that in the absence of Rsc2 the chromatin structure of the STB region is significantly altered and the Rep1 protein loses its normal localization to subnuclear foci. Rsc1, a closely related homolog of Rsc2 present in an alternative form of the RSC complex, is not required for 2m maintenance and does not replace the requirement for Rsc2 when overexpressed. This represents the first specific role for Rsc2 that has been related to a change in chromatin structure, as well as the first direct evidence linking chromatin structure to 2m segregation.The 2m circle is a high-copy-number plasmid widespread in laboratory and industrial strains of Saccharomyces cerevisiae that has been used as a molecular model for aspects of cellular function such as DNA replication, gene transcription, and recombination, as well as the basis of yeast cloning vectors. High-copy-number plasmids carrying only replication origins show maternal inheritance bias (MIB) and are poorly segregated to daughter cells (31), whereas 2m and its derivatives are effectively segregated between mother and daughter cells at division (22). However, the mechanism by which 2m escapes MIB resulting in equal segregation is not fully understood, nor have all of the host components involved been identified. Of the four genes carried by the plasmid, REP1 and REP2 are necessary for MIB to be overcome, and their products act at the plasmid locus STB to confer plasmid stability (2,10,18,22,33,36). Within the nucleus, Rep1 and Rep2 proteins are localized into discrete foci (39) in which the plasmid is also colocalized (39, 52). These plasmid and protein foci are segregated in a manner similar to that of the chromosomes themselves, a finding consistent with the observation that the 2m plasmid is packaged into nucleosomes (50). Thus, the plasmid may be exploiting the chromosomal segregation machinery indirectly by associating with specific or nonspecific chromosomal sites. It has been therefore suggested that the segregation of 2m, and hence its overcoming of MIB, likely requires host (non-plasmid-encoded) genes (18). One candidate is the product of the SHF1 (CST6) gene, which has been shown to bind to STB in vivo and in vitro (51).Nucleosomes regulate and modify the ability of proteins to interact with DNA, and the remodeling of chromatin and repositioning of nucleosomes is important in gene regulation and other functions of DNA. There is evidence that specific chromatin structures are...

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.

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.

Circular plasmids in yeast carrying only an origin of DNA replication (ARS) exhibit maternal inheritance bias (MIB) and are poorly transmitted from mother to daughter cell during division. A variety of different sequences that overcome MIB have been described, including centromeric sequences (CEN), telomere-associated repeats, silencer sequences and a specific system encoded by the endogenous 2 micron circle plasmid requiring the cis-acting locus STB and the proteins Rep1 and Rep2. In each case, DNA segregation between mother and daughter cells is dependent on DNA-protein interactions. Using plasmids carrying multiple copies of a lac repressor binding sequence, we have localised DNA molecules in the yeast nucleus using a green fluorescent protein (GFP)-lac repressor fusion protein. We compared GFP localised plasmids carrying a centromere sequence with plasmids based on 2 micron circle carrying or lacking the STB sequences required for their segregation. We show that GFP localised plasmid carrying the complete STB locus co-localises with the plasmid proteins Rep1 and Rep2 to discrete chromatin sites. These sites are distinct from both the telomeres and from sites of cohesin binding. Deletion of the region of STB essential for the stability of the plasmid, leads to a loss of plasmid association with chromatin, relocalisation of plasmids towards the nuclear periphery, and a decrease in the Rep1 protein associated with the plasmid. We conclude that specific plasmid localisation is likely to be important in the overcoming of MIB in yeast.