Linear derivatives of Saccharomyces cerevisiae chromosome III can be maintained in the absence of autonomously replicating sequence elements.

Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.

Regulation of the yeast Ace2 transcription factor during the cell cycle.

Previous studies have revealed many parallels in the cell cycle regulation of the Ace2 and Swi5 transcription factors. Although both proteins begin entry into the nucleus near the start of mitosis, here we show that Ace2 accumulates in the nucleus and binds DNA about 10 min later in the cell cycle than Swi5. We used chimeric fusions to identify the N-terminal region of Ace2 as responsible for the delay, and this same region of Ace2 was required for interaction with Cbk1, a kinase necessary for both transcriptional activation by Ace2 and asymmetric distribution of Ace2. Ace2 and Swi5 also showed differences in prevalence during the cell cycle. Swi5 is apparently degraded soon after nuclear entry, whereas constant Ace2 levels throughout the cell cycle suggest Ace2 is exported from the nucleus. Our work suggests that the precise timing of Ace2 accumulation in the nucleus involves both a nuclear export sequence and a nuclear localization signal, whose activities are regulated by phosphorylation.

ACE2, CBK1, and BUD4 in budding and cell separation.

Mutations in the RAM network genes, including CBK1, MOB2, KIC1, HYM1, and TAO3, cause defects in bud site selection, asymmetric apical growth, and mating projections. Additionally, these mutants show altered colony morphology, cell separation defects, and reduced CTS1 expression, phenotypes also seen by mutating the Ace2 transcription factor. We show that an ACE2 multicopy plasmid suppresses the latter three defects of RAM network mutations, demonstrating that Ace2 is downstream of the RAM network and suggesting that these phenotypes are caused by reduced expression of Ace2 target genes. We show that wild-type W303 strains have a bud4 mutation and that combining bud4 with either ace2 or cbk1 in haploids results in altered colony morphology. We describe a timed sedimentation assay that allows quantitation of cytokinesis defects and subtle changes in budding pattern and cell shape. Experiments examining budding patterns and sedimentation rates both show that Ace2 and Cbk1 have independent functions in addition to their common pathway in transcription of genes such as CTS1. SWI5 encodes a transcription factor paralogous to ACE2. Additive effects are seen in cbk1 swi5 strains, and we show that activation of some target genes, such as EGT2, requires either Swi5 or Ace2 with Cbk1. The relative roles and interactions of Ace2, Cbk1, and Bud4 in bud site selection, polarized growth, and cell separation are discussed.