The yeast Rad6 protein: a mediator of homologous recombination across the scaffold attached region at the replication origin ARS1.

Here we show that the ubiquitin-conjugating enzyme Rad6p plays a crucial role in locus-specific replacement recombination in the TRP1-ARS1 region. In rad6-1 strains, where this ubiquitination activity is modified, homologous recombination across a 150 bp continuous region is completely abolished. Our results unambiguously identified the ARS1 scaffold attached region (SAR) as being the region where this impediment for replacement recombination is located, since a merging of the location of the recombination impediment and binding properties in a scaffold exchange assay with deletion mutations was observed.

Replication properties of ARS1 plasmids in Saccharomyces cerevisiae: dependence on the carbon source.

The replication behaviour of a number of ARS1-based plasmids was investigated on propagation in Saccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kb TRP1-ARS1 fragment is present on a plasmid.

A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae.

We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast. This was done by substituting the chromosomal copy with a series of ars1 deletion mutants. It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains.

Mutational analysis of a variant of ARS1 from Saccharomyces cerevisiae.

A naturally occurring single base-pair G to A transition, creating a 10/11 near-match close to the essential 11 base-pair core consensus of ARS1, was used to investigate the importance of near-match sequences. The 10/11 near-match can not substitute for the core consensus since an ARS- phenotype is observed when the core consensus is deleted. However, deletion mutations revealed that this near-match together with a short palindromic sequence, also situated in the B-flanking region, comprise a single element crucial for optimal ARS function.

Maintenance and copy number control of ARS1 plasmids in Saccharomyces cerevisiae. Evidence of a mating type effect.

Following mating of a and alpha isogenic haploids we observe that the frequency of plasmid bearing cells, during selective growth, increases three fold. By examining the mitotic stability, the frequency of plasmid bearing cells during the cell cycle and the copy number of ARS1 plasmids in isogenic haploid and diploid cells, we show that the apparent stability of circular ARS1 plasmids in a/alpha cells is largely due to a diminished copy number in these cells. This observation is fully comprehensible with the model for plasmid segregation as presented by Murray and Szostak (1983).